Sorting and immobilization system for nucleic acids using synthetic binding systems

ABSTRACT

Methods are provided for producing an array of immobilized nucleic acids on an array device. The array device has a plurality of microlocations each having an electrode. At least one of the microlocations has a synthetic addressing unit coupled to the microlocation. The microlocation is the activated, usually by electronically biasing the electrode of the microlocation. The at least one microlocation is then contacted by a conjugate which has a nucleic acid and a synthetic binding unit. The conjugate is then coupled to the microlocation through an interaction between the synthetic binding unit and the synthetic addressing unit. In one embodiment, the synthetic binding unit and synthetic addressing unit may be pRNA, pDNA, or CNA.

This is a continuation of U.S. application Ser. No. 09/910,469, filedJul. 19, 2001, which is incorporated herein by reference in itsentirety.

FIELD OF INVENTION

The present invention relates to conjugates of synthetic binding unitsand nucleic acids. The present invention also relates to methods forsorting and immobilizing nucleic acids on support materials using suchconjugates by specific molecular addressing of the nucleic acidsmediated by the synthetic binding systems. Particularly, the presentinvention also relates to novel methods of utilizing conjugates ofsynthetic binding units and nucleic acids to in active electronic arraysystems to produce novel array constructs from the conjugates, and theuse of such constructs in various nucleic acid assay formats. Inaddition, the present invention relates to various novel forms of suchconjugates, improved methods of making solid phase synthesizedconjugates, and improved methods of conjugating pre-synthesizedsynthetic binding units and nucleic acids. The present invention alsorelates to the use of conjugates of synthetic binding units and nucleicacids as substrates for various enzymatic reactions, including nucleicacid amplification reactions.

BACKGROUND OF THE INVENTION

Nucleic acid chemistries and analyses have risen to a place ofprominence in such diverse areas as biological research, medicine,agriculture, and even forensic science. A key need in the use of nucleicacids in all of these areas is the ability to manipulate them on amacroscopic scale by localizing particular nucleic acid species (orgroups of species) at a known location, such as in an array on asubstrate. In order to immobilize nucleic acids on support materials, awide range of methods have been devised, which can be loosely classifiedby the stability of the bond. A covalent immobilization, i.e. animmobilization in which the nucleic acid is linked to molecularstructures of the support material by covalent bonds, is typicallyirreversible without risking the degradation of the immobilized nucleicacid. In contrast, complexing reactions, or reactions between twopartners of a binding system which specifically recognize each other,may be used. These reactions may be reversible or, for practicalpurposes, irreversible over the timescale of a particular experiment.Whether a binding system for immobilizing nucleic acids is to be denotedreversible or irreversible depends ultimately on the position of theequilibrium between bound and free nucleic acids. An example of abinding system to be regarded as practically irreversible is thecomplexing of biotin by avidin (or streptavidin, or their variousengineered equivalent proteins,) with a binding constant ofK_(a)≈2.5×10¹³ (M⁻¹) (Chilkoti, A.; Stayton, P. A.; J. Am. Chem. Soc.117, 10622-10628 (1995); Greene M.; Advances in Protein Chemistry;85-132 (1975)). This system has been widely used for immobilizingnucleic acids and other biomolecules on support materials.

WO 86/07387 describes a different example of reversible binding ofnucleic acids to surfaces. As described here, nucleic acid bindingsequences are immobilized on support materials by means of complexingagents, for example with the aid of antibody/antigen pairs.

Another example of a reversible pairing system which has been used aresets of natural-nucleotide oligomers, which specifically hybridize toprovide duplexing “tags” for immobilization. In contrast tobiotin-streptavidin, nucleic acid oligomers have an informaticdimensionality in pair formation: there are a multiplicity of specificbinding sets which may be devised, characterized by the sequence ofmonomers (nucleotides) which specifically pair only with complementarysequences. Unlike multi-hapten-lectin systems, or multi-antibody-antigensystems, such sets of nucleic acid tags have fairly uniform chemical andthermodynamic characteristics, which facilitates their use in multiplexreactions.

EP 0 305 145 describes, for example, the use of homopolynucleotide tailsand their specific pairing with complementary homopolynucleotideoligomers for immobilizing target-specific oligonucleotides. On theother hand, JP 03 151 900 describes the use of specific nucleic acidsequences for immobilizing target-specific nucleic acids. It ischaracteristic of such systems that the oligonucleotide to beimmobilized is composed of two parts: one oligomeric part complementaryto the oligonucleotide immobilized on the surface of the support, and asecond oligomeric part specific for interaction with components of thesample (e.g., complementary to a sample nucleic acid.) Similar systemsare also described in WO 93/13225, WO 93/13223, WO 93/25563, U.S. Pat.No. 5,763,175, WO 97/32999, WO 00/58516 and in WO 00/60124.

The disadvantage of the methods described above is that the sequenceused for the immobilization can potentially hybridize with the sequenceto be immobilized, forming intramolecular secondary structures, mayhybridize with another sequence to be immobilized, formingintermolecular secondary structures, or may hybridize with nucleic acidsfrom the sample. The risk of such an unwanted or interfering interactionincreases with the length of the nucleic acid(s) to be immobilized, aswell as with the complexity of a sample (e.g., the possibility ofcontaminating nucleic acids from unknown organisms.)

Another disadvantage of the use of natural nucleic acids forimmobilization is that the stability of duplexes of natural nucleicacids does not increase linearly in proportion to length (number ofnucleotides in the sequence) over a large range, but rather approaches alimit which depends only on the relative percentage of CG to AT basepairs (“CG content”). Binding systems having a duplex stabilityexceeding the natural limit cannot be prepared using natural nucleicacids. This limitation is also problematic when applying variousstringency conditions to the nucleic acid at its immobilized location:the immobilizing nucleic acid tags will also be subjected to the samestringency conditions (i.e., chaotropic agents, thermal conditions, orelectrostatic forces), and may dissociate. See G. Michael Blackburn andMichael J. Gait, eds. Nucleic Acids in Chemistry and Biology, 2nd ed.,1996, Oxford University Press, New York.

Achieving a fine differentiation in stringency differentiation betweenimmobilization tag interactions (which must remain hybridized) and thetarget-specific interactions (which are often discriminated at thesingle base pair mismatch level) is often difficult, especially underclinical-type conditions when the method must be particularly robust andconsistent.

Another significant economic and time disadvantage of using naturalnucleic acids as immobilization agents is that a certain minimumsequence length is required to reach a practical level of stability andselectivity of the immobilization. It is to typical to use 20-mers inorder to achieve sufficient binding specificity. This results in theentire nucleic acid strand (composed of the sequence for recognizing thesample and the sequence for immobilization) becoming relatively long.The use very long sequences can be disadvantageous for several reasons.First, the use of long nucleic acid sequences increases the likelihoodof secondary structure formation intramolecularly, and also increasesthe likelihood of transient or stable hybridization between multiplestrands in solution.

Active electronic array devices have been described for theelectrophoretic transport and manipulation of nucleic acids, see U.S.Pat. Nos. 6,245,508; 6,225,059; 6,051,380; and 6,017,696, the text ofeach of which is hereby incorporated by reference in their entirety.When manipulating nucleic acids on active electronic arrays, the use ofshorter sequences is preferred. Electro-kinetic addressing and movementon the electronic chip array work particularly well with relativelyshort nucleic acids because of the better electrophoretic mobility ofthe smaller molecules. Thus, shorter sequences for use as immobilizationcomplexing agents have increased utility in the context of activeelectronic arrays.

Another disadvantage of using natural systems for the immobilization ofnucleic acids is that such systems can be easily degraded or destroyedduring their use. In particular, degradation by enzymatic components ofthe sample, or even contaminating DNAses and RNAses from laboratoryworkers' fingertips, is a concern. Degradation or fragmentation byhydrolysis of the nucleic acids used for immobilization, in particularby enzymes such as, restriction enzymes, exonucleases or endonucleases,in not uncommon when a nucleic acid oligomer is allowed to sit at roomtemperature for a few days. Thus, the use of complexing agents forimmobilization which are not subject to degradation or modification bynaturally occurring enzymes is desirable.

In the course of the last few decades, a plurality of technologies havebeen developed to take advantage of the diverse natural variety ofenzymes to modify nucleic acids. Restriction endonuclease reactionsspecifically cleave nucleic acids at defined sequence sites, andnucleases or other enzymes can be utilized to degrade or modify nucleicacids at either termini. In addition, polymerases and terminaltransferases can be utilized to build nucleic acid oligomers fromnucleotides. Ligase enzymes may also be utilized to connect, or ligate,different nucleic acid strands with one another, in single-strandtemplate dependant, blunt-end double-stranded, or single-strand templateindependent manners. These enzymatic tools have become a mainstay ofanalytical biochemistry, and are necessary for almost any molecularbiology research at some point. For instance, polymerases are commonlyused for carrying out nucleic acid amplification reactions andsequencing reactions, which are both necessary components of theproduction of proteins of interest in research.

In most cases, purification steps or physical separation steps arerequired for handling at each stage of the enzymatic manipulations ofnucleic acids. Nucleic acids are usually purified by precipitation,electrophoretic separation, or chromatographic steps. For isolation andimmobilization purposes, nucleic acids are often modified with anaffinity tag, like biotin. These biotin-modified nucleic acids can bebound stably and irreversibly to solid phases via macromolecularbiotin-streptavidin complexes (e.g., DE 40 011 54 and EP 0 063 879). Thevery large complex obtained can be separated again only by rather harshchemical conditions. Although a commonly used complex, this providesonly one immobilization interaction tool: multiplex reactions withspecific localization of the products cannot be done with abiotin-streptavidin affinity system alone. Alternatively, it is possibleto elaborately incorporate polyhistidine modifications, or steroids orhaptens, such as digoxigenin, into a nucleic acid in order to makeseparation possible by fixing to binding partners corresponding to themodifications. However, these systems only allow separation underdiverse conditions (nickel chromatography, vs antibody binding,) andthus do not overcome the limitations of the biotin-avidin interactionfor multiplex reactions.

Chemical conjugation of one nucleic acid to another nucleic acid insolution requires that one nucleic acid is provided with a modificationwhich can react with the modification of the other nucleic acid byforming a stable bond. Often, finished, pre-synthesized nucleic acidsare conjugated with other nucleic acids and analogs by utilizing theability of nucleic acids to form complementary pairs with themselves, orby the pairing of the nucleic acids with a nucleic acid template forligation. The pairing leads to an association or pre-organization of theparts to be conjugated, which supports or else makes thermodynamicallyfavorable the subsequent conjugation reaction. For example, NucleicAcids Research 16(9), 3671-3691 (1988) describes the conjugation ofthiol-modified nucleic acids. This option allows, when treated withatmospheric oxygen, the formation of both homodimers and heterodimers,and is therefore only provisionally suitable for linking together twodifferent nucleic acids. The template-supported conjugation ofaldehyde-modified nucleic acids with amine-modified nucleic acids isdescribed in J. Am. Chem. Soc. 114, 9197-9198, (1992). Anothertemplate-supported photochemical conjugation is described in NucleicAcids Research 26(13), 3300-3304, (1998). One of the few reactionsdescribed without support by self-binding or template binding is thereaction of phosphorothioates with α-haloacetylene (Gryaznov S M, J. Am.Chem. Soc. 115, 3808-3809, (1993)). WO 01/07657 describes the linkage ofRNA building blocks at the 3′ end of an RNA oligonucleotide by oxidationwith periodate to give the dialdehyde and subsequent reaction with anitrogen nucleophile to give a cyclic product. The nitrogen nucleophilesused may be amines, hydrazines, hydrazides, semicarbazides orthiosemicarbazides. The product formed initially can be stabilized byreduction with NaCNBH₃.

Since synthetic binding systems, such as pyranosyl-RNA (pRNA) orpyranosyl-DNA (pDNA) binding systems, are, by design, not stericallycapable of pairing with nucleic acids, these previously describedmethods are not applicable to conjugating nucleic acids with syntheticbinding units. However, the solid-phase tandem synthesis of syntheticbinding unit/nucleic acid conjugates by phosphoramidite chemistry is notdesirable for circumstances in which a nucleic acid is readily availablefor conjugation (e.g., a bacterial plasmid preparation.) Likewise, ifthe nucleic acid to be conjugated is longer than about 20 nucleotides,the size of the conjugate (nucleic acid+6-15 pRNA residues) approachesand eventually surpasses the efficient synthesis limit of thesolid-phase chemistry. There is therefore a need to find methods andconditions which make it possible to conjugate finished, pre-synthesizednucleic acids with synthetic binding systems.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides novel methods for the activeconstruction of arrays of immobilized nucleic acids utilizing activearray devices. In the invention, an array of immobilized nucleic acidsis produced on an active location array device comprising a plurality ofactivatable locations on a support material, by:

-   -   a) activating a first set L₁ of locations on the active array        device, wherein at least some of the activated locations        comprise, or have attached thereto, a predetermined synthetic        addressing unit (SAU) or set of synthetic addressing units        attached at the locations, and wherein the activation of the        locations creates a condition favorable to the binding of the        SAUs to SBUs;    -   b) contacting the activated set of locations with a first set C₁        of synthetic binding unit (SBU)-nucleic acid (NA) conjugates,        wherein at least some of the SBUs in the set of conjugates are        capable of specifically binding to at least some of the SAUs        attached at the activated locations;    -   c) removing the unbound conjugates; and    -   d) repeating steps (a) through (c) M number of times, activating        set L_(M+1) of locations, and contacting them with set C_(M+1)        of conjugates, wherein M≧1.

In embodiments of the first aspect, it is preferred that at least tolocations of the active device, the same SAU is attached. Preferredembodiments also utilize rectangularly arrayed locations. Particularlypreferred embodiments may use active electronic array devices,optionally with electronic washing. The various specific SAUs and SBUsdescribed throughout the specification are useful in this first aspect,as are the various specific conjugate structures. Likewise, the variouslibraries described throughout the specification may be used as sets Cin this aspect of the invention. In various embodiments of the firstaspect of the invention, the SAUs may be attached to the locations ofthe array by active addressing means, or passively by more traditionalspotting means.

In various embodiments of the first aspect of the invention, at least10, at least 100, at least 1000, or even 10,000 or more distinct nucleicacids may be immobilized on the active location array. Likewise, in theinvention, various numbers of conjugates in each set C may beimmobilized at each cycle M, such as groups of at least 5, at least 10,at least 20, or even 100 conjugates at each cycle. This may be carriedout for several cycles, preferably where M is between 1 and 100,preferably between 1 and 20, or also preferably between 1 and 10,depending on the size of the active location array, and the desirednumber of conjugates in each set C.

In additional embodiments of this first aspect, the conjugates forimmobilization on the array are first contacted with a sample,preferably a biological sample, before being contacted with the solidsupport. In preferred embodiments, the conjugates may be utilized in anamplification reaction while contacted with the sample. In otheradditional embodiments, the constructed array is later contacted with asample, preferably a biological sample. Optionally, the array may besubjected to a detection step, in which the immobilized conjugates, ortarget nucleic acids hybridized to the immobilized conjugates, aredetected.

In additional embodiments of the first aspect of the invention, thearray may be subjected to a further step in which the conjugates areremoved from the array by disrupting the SBU/SAU interaction, thusregenerating the support with attached SAUs for further use.

A second aspect of the invention are novel array constructs, which areproduced using the active array construction methods. These novelsupermolecular constructs comprise:

-   -   a) at least one synthetic address unit (SAU) (either a single        SAU or a mixture of SAUs) attached to a support material        comprising an array of discreet locations, wherein the same SAU        is attached to at least two predetermined locations on the        support material, and    -   b) at least two conjugates comprising a synthetic binding unit        (SBU) and a nucleic acid (NA), wherein at least two of the        conjugates have the same SBU and different NAs,    -   c) wherein the SBU of the conjugates form a synthetic binding        system unit (SBS) with the SAU at the two predetermined        locations, and immobilize each of the two different NAs at a        different location.

The SAUs and SBU conjugates described throughout the application may beutilized in various embodiments of the second aspect of the invention.In addition, various types of support materials described below may beutilized. A preferred embodiment of the second aspect utilizes an activeelectronic array as the support for the supermolecular construct. Thesupermolecular constructs may comprise at least 5, at least 10, or 100or more different conjugates immobilized by the same synthetic bindingsystem. The constructs may also comprise at least 10, at least 100, atleast 1000, or even 10,000 or more different immobilized nucleic acids.in preferred embodiments of the second aspect, the supermolecularconstruct comprises two or more different SAUs attached to differentlocations on the support material, where the SAUs are orthogonal.

In a third aspect, the invention provides several novel conjugates whichcan be used to sort and immobilize nucleic acids on a support materials.The conjugates of this aspect of the invention have the general formula(NA)(SBU) wherein:

-   -   NA is a nucleic acid,    -   SBU is a synthetic binding unit,        wherein each NA is linked to at least one SBU by a linker X,        wherein X is selected from the group consisting of:        wherein    -   Y₁,Y₂ are independently of one another OH; SH, NH₂ or CH₃,    -   G is O or S,    -   L₁, L₂ are independently linkers selected from the group        consisting of:        -   a covalent bond; and a linker chain moiety comprising a            saturated or unsaturated, branched or unbranched,            substituted or unsubstituted, chain of 1-60 carbon atoms and            0-40 heteroatoms selected from the group consisting of N, O,            and S;    -   V₁, V₂ are independently selected from the group consisting of    -   R₁, R₂ are independently of one another H or OH, and wherein    -   Ba is a nitrogen heterocycle moiety.

In various embodiments of this aspect of the invention, the linker X mayhave any of the general formulae (i)-(v), and may be oriented in eitherdirection. In various embodiments of the invention, L may be linkersincluding:—[—(CH₂)_(n)—]—or —[—CH₂—CH₂—(O—CH₂—CH₂)_(m)]—or —[—CH₂—CH₂—CH₂—(O—CH₂CH₂—CH₂)_(q)]—or —[—(CH₂)_(v)—C(O)NH—(CH₂)_(z)—]—or —[—(CH₂)_(v)—NHC(O)—(CH₂)_(z)—]—with n, m, q, v, z in each case independently of one another being aninteger between 1 and 20, and more preferably, n is between 2 and 12, mis between 1 and 5, q is between 1 and 4, and v and z are independentlybetween 2 and 6.

In a fourth aspect, the invention provides novel branched or linearconjugates, with the general formula (NA)_(n)(SBU)_(m), wherein:

-   -   NA is a nucleic acid,    -   SBU is a synthetic binding unit,        wherein each NA is linked to at least one SBU, n is an integer        from 1 to 6, m is an integer from 1 to 6, and wherein n+m>2. In        preferred embodiments of the fourth aspect of the invention, the        conjugates have a structure with a general formula selected from        the group consisting of (I), (II), or (III):        (NA)-X₁—(SBU)—X₂—(NA)   (formula (I))        (SBU)—X₁—(NA)-X₂—(SBU)   (formula (II)),          (NA)-W—(—(SBU))_(u)   (formula (III)),        where u is an integer between 2 and 6.

In various embodiments of the fourth aspect of the invention, NA iscovalently linked to at least one SBU via a linker moiety X or abranching moiety W, wherein X, independently for each linker unit, isselected from the group consisting of:

wherein

-   -   Y₁,Y₂ are independently of one another OH, SH, NH₂ or CH₃,    -   G is O or S,    -   L₁, L₂ are independently linkers selected from the group        consisting of:        -   a covalent bond; and a linker chain moiety comprising a            saturated or unsaturated, branched or unbranched,            substituted or unsubstituted, chain of 1-60 carbon atoms and            0-40 hetero atoms selected from the group consisting of N,            O, and S;    -   V₁, V₂ are independently selected from the group consisting of    -   R₁, R₂ are independently of one another H or OH, and    -   Ba is a nitrogen heterocycle moiety;    -   and wherein W has the general formula:    -   wherein Y₁ is OH, SH, NH₂ or CH₃;    -   wherein D₁, D₂, D₃, and D₄, are, independently, a covalent bond        or a linker chain moiety comprising a saturated or unsaturated,        branched or unbranched, substituted or unsubstituted, chain of 1        to 10 carbon atoms and 0 to 4 heteroatoms selected from the        group consisting of O, S, and N;    -   wherein J is carbon or nitrogen;    -   wherein z is 0 or 1, further wherein z is 0 if J is nitrogen;        and    -   wherein X₁, X₂, and X₃, are independently X as described above.

In preferred -embodiments of the fourth aspect of the invention, W hasone of the following general structures:

The SBUs and NAs for use in the third and fourth aspects of theinvention may be any of those described herein. Preferred SBUs includepRNA, pDNA, and CNA. Preferred NAs include DNA, RNA, and theirchemically modified derivatives. Labeled embodiments are also preferred.

In a fifth aspect, the present invention provides unit of a syntheticbinding system selected from the group consisting of a synthetic bindingunit (SBU) and a synthetic addressing unit (SAU), comprising an oligomerof monomeric units, wherein the monomeric units have the general formula

wherein B_(b) is a backbone moiety which connects the monomeric unit tothe oligomer, and wherein R^(s) is a specific recognition moietycomprising a nitrogen heterocycle moiety, wherein the monomeric unitslinearly arranged according to the formulae (IX) or (X),B_(s1)-(J)-B_(s2)   (formula (IX))B_(s1)-(J)-B_(s2)-(J′)-B_(s3)   (formula (X))wherein s1, s2 and s3 are, independently, an integer between 0 and 10,and B is any monomeric building block as is used for synthesizing thesynthetic binding units (SBU), and J is a sequence of recognitionmoieties (R^(s)), wherein J may be the same or different than J′, andwherein the sequences J and J′ are, independently, selected from group Aconsisting of SEQ. ID Nos. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, and 75, or are selected from the group Bconsisting of SEQ. ID Nos. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, and 76, provided that sequences J and J′ areselected from the same group.

In preferred embodiments of the fifth aspect of the invention, J and J′are, independently, selected from group A′ consisting of SEQ. ID Nos. 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, and 47, or are selected from the group B′ consisting ofsequences SEQ. ID Nos. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, and 48, provided that sequences Jand J′ are selected from the same group. In additional preferredembodiments of the fifth aspect of the invention, J and J′ are,independently, selected from group A″ consisting of SEQ. ID Nos. 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, or are selected fromthe group B″ consisting of sequences SEQ. ID Nos. 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, and 76, provided that sequences J and J′are selected from the same group. In more preferred embodiments of thefifth aspect of the invention, J and J′ are, independently, selectedfrom group A′″ consisting of SEQ. ID Nos. 49, 51, 53, 57, 59, 61, 65,69, 71, and 75, or are selected from the group B′″ consisting ofsequences SEQ. ID Nos. 50, 52, 54, 58, 60, 62, 66, 70, 72, and 76,provided that sequences J and J′ are selected from the same group.

In preferred embodiments, the SAUs or SBUs are pRNA, pDNA, or CNA. Invarious embodiments, the fifth aspect of the invention may be sets,libraries, or kits comprising SAUs or SBUs with the disclosed sequences,preferably where the SAUs or SBUs in the sets, libraries, or kits areorthogonal to each other. For instance, kit embodiments may comprise twoor more, more preferably at least 5, or at least 10, or at least 20, ofthe SAUs which are either unmodified or are adapted with functionalgroups for attachment to a support material. Furthermore, an SAU kitembodiment may comprise the SAUs pre-attached to a support material. Inthese embodiments preferably between 1 and 100, or between 2 and 20, orbetween 2 and 10 SAUs are attached to the support at known locations.Or, kit embodiments may comprise two or more, more preferably at leastfive, or at least 10, or at least 20 of the SBUs which are eitherunmodified or adapted with functional groups for attachment to NAs. Inaddition, the SBUs of the fifth aspect of the invention may be utilizedin any of the conjugate structures disclosed herein.

In various embodiments of the third, fourth aspects, and conjugate fifthaspects of the invention, libraries may be constructed from severalconjugates. The SBU members of the libraries are preferably orthogonal.Libraries comprise at least two, preferably at least five, at least ten,at least twenty, or at least 100 conjugates, depending on the purpose ofthe library (e.g., a library of conjugate primers for amplifying a setof genetic loci may have 20 conjugates, while a library for theproduction of a SNP or STR assay array may have over 100 conjugates). Inthe libraries, each NA may be conjugated to a specific SBU, each SBU maybe conjugated to a specific NA, or both. Alternatively, each NA may beconjugated to a set of SBUs, or each SBU may be conjugated to a set ofNAs.

In various embodiments of the third, fourth aspects, and conjugate fifthaspects of the invention, supermolecular constructs of immobilizednucleic acids in an array may be constructed from one or moreconjugates. These may use any of the SAUs described herein on any of thesupport materials described herein. Preferred embodiments use an activeelectronic array as a support. Preferred supermolecular constructscomprise at least five, more preferably at least ten, more preferably atleast 100 or at least 1000 conjugates.

In further embodiments of these aspects of the invention, the conjugatesare contacted with attached SAUs to form supermolecular constructs onsupport materials. These methods may be carried out passively oractively (e.g., by electronic addressing on an active electronic arraydevice in preferred embodiments.) In these method-embodiments, theconjugates may be contacted with a sample, preferably a biologicalsample, either prior to or after contacted with the attached SAUs.

In a sixth aspect of the invention, kits are provided for producing theconjugates of the third and fourth aspect of the invention. These kitscomprise one or more SBUs, more preferably at least 5, or at least 10,or at least 20, which have been modified with functional groups forreaction with modified or unmodified nucleic acids to produce theconjugates of the third or fourth aspect of the invention.

In a seventh aspect, the invention provides improved methods for thesolid-phase synthesis of phosphate or phosphoramidite moiety linkedconjugates. These methods comprise:

-   -   a) synthesizing the conjugates on a solid support phase using        monomer or oligomer units, wherein the units are        β-cyanoethyl-protected on a terminal phosphorus of the units,    -   b) treating the support with a solution of an alkylamine in an        inert solvent,    -   c) treating the support with hydrazine to cleave off and        deprotect the conjugate.

In preferred embodiments, the alkylamine is a secondary alkylamine, morepreferably selected from the group consisting of dimethylamine,diethylamine, dipropylamine, dibutylamine, dipentylamine, dihexylamine,di-N-octylamine, di-N-decylamine, didodecylamine, N-ethylmethylamine,N-methyl-N-propylamine, N-methylbutylamine, N-methylpentylamine,N-methylhexylamine, N-ethylpropylamine, N-(N-butyl)-N-propylamine,N-amyl-N-butylamine, N,N′-di-N-butyl-1,6-hexanediamine,N,N′-dimethyl-1,3-propanediamine, N,N′-dimethyl-1,6-hexanediamine. Mostpreferred for use is diethylamine.

In an eighth aspect, the present invention provides methods forutilizing the nucleic acid portion of a SBU—NA conjugate, as any of thestructures disclosed herein, as a substrate in an enzymatic reaction. Inone group of embodiments, the nucleic acid portion of the conjugates isenzymatically modified. In general, these methods comprise:

-   -   a) contacting the conjugate with at least one enzyme which        utilizes naturally occurring nucleic acids as a substrate, and        with other reagents necessary for the action of the enzyme; and    -   b) incubating the mixture obtained in a) under conditions        suitable for the functioning of the enzyme for a period of time        sufficient to effect the modification of the conjugate.

In various embodiments, various reagents may be utilized in step a),including nucleoside triphosphates (e.g., plain, labeled, chemicallymodified) and/or template or target nucleic acids (e.g., plain, labeled,chemically modified). In various embodiments, more than one enzyme maybe employed in step a). In one preferred embodiment, the enzyme is aligase enzyme, and the nucleic acid portion of the conjugate is ligatedto a target nucleic acid. In another preferred embodiment, the enzyme isat least one polymerase, and a template nucleic acid sequence isamplified using the conjugate as a primer. In a particularly preferredversion of this embodiment, the amplification is a linear or exponentialthermal cycling amplification. In another preferred embodiment, theenzyme is a mixture of enzymes comprising a restriction endonucleaseactivity and a polymerase activity, and a template nucleic acid sequenceis amplified isothermally utilizing a conjugate as a primer. Inparticularly preferred versions of this embodiment, the conjugate isimmobilized, and the amplification is an anchored SDA reaction.

In other embodiments of the eight aspect of the invention, the conjugateis utilized as a substrate hybridized to a target nucleic acid, whereinthe nucleic acid hybridized to the conjugate is modified by the enzyme.Preferred embodiments include the degradation of an target RNA strand byRNAse H while hybridized to a conjugate, and the cleavage of a targetnucleic acid strand by a restriction endonuclease while hybridized to aconjugate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A diagrammatic representation of some preferred arrangements forlinking nucleic acids (NA) to synthetic binding units (SBU,) and howsuch conjugates can be used for immobilization on support materials (SM)using synthetic addressing units (SAU.) FIG. 1A shows the linkage of anucleic acid via its 3′ end to the end of the SBU which points away fromthe support when paired with the SAU. FIG. 1B shows the linkage of anucleic acid via its 5′ end to the end of the SBU which points away fromthe support. FIG. 1C shows the linkage of a nucleic acid via its 5′ endto the end of the binding unit which points towards the support. FIG. 1Dshows the linkage of a nucleic acid via its 5′ end to a position in thecenter of the binding unit. An example of this type of linkage would bea the conjugation of a tryptamine base linker in a pRNA with an aldehydeon the end of a nucleic acid.

FIG. 2: An illustration of some preferred linkage arrangements andspacers for use with nucleic acids and pRNA synthetic binding units.R1═H, OH, OMe, Me; R2═H, OH, OMe; L=(CH₂)_(n);CH₂—CH₂—(—O—CH₂—CH₂—)_(m)—O—CH₂—CH₂.CH₂—CH₂—CH₂—(—O—CH₂—CH₂—CH₂—)_(q)—O—CH₂—CH₂—CH₂;Ba=heterocyclic base (C,T, U, G, A, etc.)A: Linkage of the 4′ end of a pyranosyl building blockof a SBU to the 3′ end of a nucleic acid directly through a phosphate.B: Linkage of the 4′ end of a pyranosyl building block of an SBU to the5′ end of a nucleic acid directly through a phosphate. C: Linkage of the2′ end of a pyranosyl building block of an SBU to the 5′ end of anucleic acid directly through a phosphate. D: Linkage of the 4′ end of apyranosyl building block of an SBU to the 3′ end of a nucleic acid via alinker (L). E: Linkage of the 4′ end of a pyranosyl building block of anSBU to the 5′ end of a nucleic acid via a linker (L). F: Linkage of the2′ end of a pyranosyl building block of an SBU to the 5′ end of anucleic acid via a linker (L).

FIG. 3: A diagrammatic representation of the three dimensionalstructural difference between nucleic acids and the preferred syntheticbinding systems for use in the invention. FIG. 3A shows the helical formof nucleic acid duplexes on the basis of a B-DNA helix. FIG. 3B showsthe non-helical nature of synthetic binding systems such as pRNA, pDNA,and CNA. FIG. 3C shows the example of a binding unit (SBU) 102a andaddress unit (SAU) 102b. NA=nucleic acid; SM=support material; in thisexample, the address 102b is immobilized and 102a is conjugated with thenucleic acid (NA) via a linker. The inter-strand base stackinginteractions (stacks) are shown using the example of purine-purine basestacking interactions (Pu—Pu stack) and of purine-pyrimidine basestacking interactions (Pu—Py stack). 0 stack means the absence of such abase stacking interaction. The pyrimidine-purine stacks possible inprinciple are not present in this specific example.

FIG. 4: An exemplary illustration of some embodiments of pRNA bindingaddresses and conjugates. 4A shows a synthetic address unit (SAU) havinga 4′ end biotin (BIO) for immobilization on support materials coatedwith streptavidin, 4B shows a synthetic address unit (SAU) with atetrahydrazide (4HY) moiety for immobilization on active ester surfaces,and 4C shows a conjugate of a pRNA unit (SBU) and a DNA (NA).

FIG. 5: An illustration of the conjugation of a DNA having a 3′ terminalribonucleotide (NA) with a vicinal diol, and a 2′ Cy3-labeled and 4′monohydrazide-modified pRNA (L HY pRNA) to give a conjugate (C) asdescribed in Example 1.4.

FIG. 6: An illustration of the conjugation of a 3′ glyceryl-modified DNA(Gly NA) via the oxidized aldehyde DNA (OB NA) intermediate with a 2′Cy3-labeled and 4′ monohydrazide-modified pRNA (L HY pRNA) to give aconjugate (C) as described in Example 1.5.

FIG. 7: FIG. 7A illustrates the immobilization of nucleic acids (NA)which are linked via a branching site (BS) to a plurality of identicalbinding units for the immobilization on support materials (SM). FIG. 7Bshows the use of two different synthetic binding systems (SAU1 and SBU1and also SAU2 and SBU2) for the immobilization of a nucleic acid througha branching structure. An advantage of these structures in theincreased, additive binding energy immobilizing the nucleic acid.

FIG. 8: An illustrative conjugate in which a nucleic acid (NA) is linkedvia a branching site (BS), or a branching moiety W, to a plurality ofidentical binding units (SBU).

FIG. 9: A diagram of some possible embodiments of labeled syntheticbinding addresses and synthetic binding units or conjugates. 9A shows aconjugate of a nucleic acid (NA) and synthetic binding unit (SBU) with amarker at the end of the binding unit, as through the end phosphate(SM=support material). B depicts the arrangement for the marker group(M) to be located at any position in the conjugate, for example throughthe use of a base-labeling moiety. C shows the use of a labeledsynthetic binding address. D shows the joint use of two marker groups(M1 and M2) which have been adjusted to one another and give a signal inthe form of fluorescence quenching or fluorescence resonance energytransfer (FRET) when a conjugate binds to the synthetic binding address.

FIG. 10: An illustration of an additional embodiment in which aplurality of different molecular species or nucleic acids (NA1 and NA2)are immobilized next to one another on the same array position, orcapture site. This can be achieved, as depicted in 10A, by fixing twodifferent SAU's to one site to form two different binding systems (SBS)at one array location (L). Or, as depicted in B, the same effect may beachieved by using the same binding system (SBS), and using conjugates ofdifferent nucleic acids (NA 1 and NA 2) with the same SBU.

FIG. 11: An illustration of the use of synthetic binding addresses andconjugates of synthetic binding units with nucleic acids for theconstruction of arrays on support materials (SM). Figure A shows anembodiment in which different nucleic acids (NA 1, NA 2, NA 3, . . . )are immobilized on different locations (L) of the support material usingdifferent binding units (SBU 1, SBU 2, SBU 3, . . . ) and differentaddress units (SAU 1, SAU 2, SAU 3 . . . ). The SAUs of the locationsare, for example, simply spotted on passive arrays, or may beelectronically addressed to capture sites above electrodes in the caseof active electronic arrays. A characteristic of the embodiment is thateach array location carries a distinctive binding address. Although thisis illustrated here as a separate SAU for each SP, the distinctiveaddresses may as easily be formed by mixtures of SAU's (e.g., SAU 1&2 atL 1, SAU 3&4 at L 2, etc.). FIG. 11B shows that for immobilizing anucleic acid (NA) on a support material (SM) SBU (here pRNA) and SAU(here CNA) need not belong to one molecular class. Any stericallycompatible molecules which form a stable and selective immobilizationupon recognition are suitable for use in these formats.

FIG. 12: A diagram showing an embodiment of the method for usingsynthetic address units and conjugates of synthetic binding units withnucleic acids to construct arrays on support materials. As illustrated,individual array positions can specifically and independently of oneanother be addressed by utilizing array surfaces with individuallyactivatable locations, such as, for example, active electronic arrays.Initially, N different binding addresses (a, b, c, . . . N) are fixed tothe surface of the support material. In this step, synthetic addressunits are affixed to groups of M-number of active sites utilizing thesame immobilization method (e.g., biotin-streptavidin interaction, orhydrazide-active ester chemistry) in a number of steps (e.g., spottingor electronic addressing.) The immobilization is preferably carried outso that identical synthetic binding units are deposited either in rowsor columns, although other geometries (e.g., squares, crosses, circles,or even randomly chosen groups of sites) may be used. Thus an array of Ndifferent binding addresses is obtained, in which each unit appears inM-fold repetition (FIG. 12A). In the following steps, conjugates ofnucleic acids and synthetic binding units are contacted with the arraygenerated previously. Although this can be carried out eitherindividually using individual SBU/NA conjugates, sets of differentSBU/NA conjugates are advantageously used to immobilize up to Ndifferent SBU-encoded nucleic acids in one step. It is important thatthe method can only be used in those arrays in which individual arraypositions can be activated specifically and independently of oneanother, e.g., in active electronic arrays. The activated positions ineach step create a condition which is favorable to the binding of theSBUs with the attached SAUs. In the preferred embodiment, mixtures of Ndifferent conjugates (or SBU-encoded groups of conjugates) are addressedto N array positions, and this addressing can be carried outsequentially or parallel to all N positions. These sets may contain Ndifferent nucleic acids (v, w, x, y, . . . N) all of which areconjugated with an individual synthetic binding unit (a′, b′, c′, . . .N). The specific recognition of the fixed synthetic address unit by thebinding units leads to only that type of nucleic acid, which has beenconjugated with the corresponding synthetic binding unit complementaryto the fixed binding unit, being immobilized on each array position. Inother words, the set of conjugates is sorted to specific locations.Thus, in a single process step N different conjugates (a′-v1, b′-w1,c′-x1, d′-y1, . . . N) are immobilized. Although a row/column addressingscheme is shown here, other geometries are possible. After the bindingof each set of conjugates to each activated set of locations, theunbound conjugates are removed. This procedure can then be repeatedusing a new second set of conjugates (a′-v2, b′-w2, c′-x2, d′-y2, . . .N). In each repetition, again up to N conjugates are immobilized inparallel. Thus, after M repetitions, W different conjugates areimmobilized on the array, with W=N×M.

FIG. 13: shows further embodiments for using synthetic binding systemsfor the immobilization of nucleic acids on support material. FIG. 13Ashows an example of an array of N different address units. In additionalembodiments, one may wish to attach individual address units two or moretimes to different array positions for duplicate addressing at thosepositions. This embodiment is typical for passive arrays, although itmay be utilized in active location array systems as well. FIG. 13Bshows, by way of example, an embodiment for using synthetic bindingsystems for generating arrays of nucleic acids for the analyses ofdifferent samples of nucleic acids, as can be applied to gene expressionstudies. Here, different times, stages or populations of nucleic acids,for example particular gene probe sequences which have been hybridizedto different samples, are compared with one another on an array. Here,by way of example, the immobilization of three times T1, T2, T3 ofvarious nucleic acid sequences (genes; S1, S2, S3, . . . ) is depicted.Initially, an array of fixed synthetic address units is prepared. Timesto be compared may thereafter by differentiated by particular sets ofSBU's conjugated with the set of nucleic acid sequences to be monitored.The times to be compared of may be allowed to associate with the SAU'seither simultaneously or sequentially. The specific recognition ofsynthetic address units by the synthetic binding units immobilizes oneach array position only the conjugate specifically belonging to theposition, thus locating the sample nucleic acids hybridized according tothe SBU utilized. An advantage to simultaneous addressing of severalsamples at one once is that variability in hybridization conditionscaused by the individual incubation of the samples with the array may beavoided, allowing for more rigorous and quantitative comparison of theresults. In addition, the ability to assay multiple samples in one cyclesaves time compared with individual addressing, or hybridization withseparate arrays.

FIG. 14: A graph of the result of a surface plasmon resonance experiment(SPR) in which 14 synthetic address units are contacted with14-synthetic binding units. The signal increasing from the baselineupward indicates binding. As expected, all address units bind to the ineach case corresponding specific binding unit (e.g. 102a to 102b). Someaddress units and binding units, however, show some degree ofcross-association with units which are not exactly complementary (e.g.105b with 110a). Sets of binding units in which such cross-associationreactions are substantially reduced are said to be “orthogonal”.Although suitable for some uses, non-orthogonal units should not be usedtogether in one set of synthetic binding systems when strict sortingaccording to the addressing units is desired.

FIG. 15: A photograph and chart of the specific association offluorescently labeled binding units to synthetic address units attachedto the surface of an active electronic array. The strongest fluorescencesignals are all on a diagonal with the specific SBU—SAU pairs, asexpected.

FIG. 16: A chart of the association of conjugates of nucleic acids (C)and pRNA/SBUs with pRNA/SAU's attached to the surface of the SPR chip,also demonstrating specific binding between the coordinating pairs. 4′biotinylated 102b, 103b, 104b, and 105b, prepared as in Example 2.1,were immobilized on the Sensor Chip channels as described in Example 8.Conjugates IL4RP102a, IL4RP103a, and IL4RP104a, prepared as in Example1.1, were then sequentially assayed for binding to the SAUs in thechannels.

FIG. 17: A chart of the immobilization of a conjugate IL4RP102a (Example1.1) by biotinylated synthetic address unit 102b (Example 2.1) on an SPRchip and subsequent hybridization of the nucleic acid part of theconjugate with three complementary DNA fragments. IL4RP102C1 was acomplementary sequence to all but the 5′ three nucleotides of the NAportion of IL4RP102a, IL4RP102C2 was a full complementary sequence, andIL4RP102C3 was a full complementary sequence plus three nucleotides.This experiment also illustrates the ability of the attached SAUs to beutilized repeatedly by simply stripping of the bound SBU conjugates witha mild base solution (e.g. 10 mM NaOH).

FIG. 18: A diagram of the setup and negative photographs of the resultsof the Example 5.2. Mixtures of cy3 labeled and one unlabeled syntheticbinding unit-nucleic acid conjugates (SBU—NA) are addressed on an activeelectronic array. Each conjugate is immobilized to the position with thematching synthetic address unit (SAU). The fluorescence measured isdisplayed as a diagram and a chip image.

FIG. 19: 19A: a diagram of the use of a conjugate (C) of a syntheticbinding unit (SBU) and a nucleic acid section (NA) as the substrate(primer) for a polymerase (E) which carries out a nucleic acidtemplate-dependent synthesis, a first step in amplification reactions.19B: a diagram of the use of a conjugate (C) of a synthetic binding unit(SBU) and a nucleic acid section (NA) as the substrate of asingle-strand ligase (E) which links the conjugate to a nucleic acid.Although shown with a relatively long NA section on the conjugate,ligase reactions may utilize conjugates with just a single nucleic acidresidue.

FIG. 20A & B: Diagrams of the use of a conjugate (C) of a syntheticbinding unit (SBU) and a nucleic acid section (NA) as substrates fornuclease reactions. A: The conjugate and a target nucleic acid arecleaved in a restriction endonuclease (E) reaction. This reaction can beespecially useful when coupled with fluorescent energy transferlabeling, as shown here. The emissions of a fluorophore moiety (F) maybe masked by placing a quencher moiety (Q) on the same conjugate. Aftercleavage, emissions of the fluorophore are no longer absorbed by thequencher, and become visible. The progress of this reaction may bequantified dynamically (real time), or after completion, in order toquantify the amount of the target in the sample. B: The target nucleicacid is selectively degraded by an endonuclease (E) when hybridized tothe conjugate nucleic acid portion. This can be especially useful fordegrading RNA target nucleic acids with double-stranded specific RNAses,such as RNAse H.

FIG. 21: An illustration of representative pRNA-DNA conjugates. A: aNA/SBU conjugate of a pRNA (SBU) with 2′-OMe-RNA (NA) and 5′ phosphate(P) (a suitable substrate for a ligase reaction); the conjugateadditionally carries a fluorescence dye at its 3′ end. B: a NA/SBUconjugate of a pRNA unit (SBU) and a DNA (NA) with free 3′ end (asuitable substrate for a polymerase or restriction endonucleasereaction.)

FIG. 22: A: a photograph of an agarose gel showing the formation ofspecific of amplification products in a PCR reaction using pRNA-DNAconjugates as primers. For comparison, mixtures using standard DNAprimers were also applied (see Example 2). B: another photograph of anagarose gel showing the result of a large-scale PCR reaction usingNA/SBU conjugates as primers (see Example 3).

FIG. 23: A photograph of a 10% polyacrylamide gel showing the productsof ligation of a fluorescence-labeled conjugate of pRNA and 2′-OMe-RNAwith a target RNA, under both UV shadowing and fluorescence imaging. Dueto its short length, the free unligated NA/SBU conjugate is notdisplayed on the gel (see Example 4). The Lanes of the gels are asfollows:

-   -   Lane 1: Acceptor RNA (negative control)    -   Lane 2: Ligation mixture with 100 pmol of acceptor RNA and 300        pmol of pRNA hybrid    -   Lane 3: Ligation mixture with 100 pmol of acceptor RNA and 1000        pmol of pRNA hybrid    -   Lane 4: Ligation mixture with 100 pmol of acceptor RNA and 300        pmol of pRNA hybrid    -   Lane 5: Ligation mixture with 100 pmol of acceptor RNA and 1000        pmol of pRNA hybrid

FIG. 24 An illustration showing, schematically, an array of SAU onlocations (L) to which a series of distinct SDA primers have beenaddressed via the interaction between the SAU and SAU.

FIG. 25: An illustration showing that that two different primers can beaddressed using a single SBS by using a branched linker to attach theprimers to one SBU.

FIG. 26: An illustration showing that shows the general architecture ofthe SBU anchored SDA construct. The SDA primers contain a restrictionsite template and a region complementary to the target DNA. The bumperprimers are sequences complementary to a region of the target DNAupstream of where the SDA primer binds. The bumper primers form theinitiation sites for the polymerase and lead to the initial stranddisplacement of the target nucleic acid from the extended primer.

FIGS. 27A and 27B: An illustration of the initiation steps forSBU-anchored SDA (phase 1):

Copying of the target sequence onto the SBU-anchored SDA primer,Displacement of the genomic DNA by extension from bumper primer 1,Activation of the restriction site, and Generation of displaced S1strands.

FIG. 27C: An illustration of the linear amplification reactions of SBUanchored SDA (phase 2): The generation of a single stranded anchoredamplicon for every Phase 1 displaced strand captured.

FIGS. 27D and 27E: An illustration of the exponential amplificationreactions of SBU anchored SDA (phase 3): Activation of restriction sitesin both anchored amplicons, Generation of displaced S1 and S2 strands,and exponential amplification via bridging and capture.

FIG. 28: An illustration of the specific addressing experiment describedin Example 5.3. The first two columns of addressed, immobilized nucleicacid/SBU conjugates and hybridized labeled nucleic acids are illustratedby the small figures under the headings “Column 1” and “Column 2”. Thegreen fluorescent signal is shown to the left, and the red fluorescentsignal is shown to the right, for each of the two columns. Note thatthis experiment demonstrates that there is no significant hybridizationof the complementary sequences to non-activated locations within thearray, allowing one to effectively isolate hybridization reactions usingthe same sequences from different samples at different locations.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have demonstrated a number of improvements in the productionand use conjugates comprising at least one nucleic acid (NA) and atleast one synthetic binding unit (SBU), wherein the synthetic bindingunit is able to specifically recognize a synthetic molecular addressunit (SAU) attached to a surface. The specific recognition of SBUs bySAUs can lead to constructs which include a synthetic binding system(SBS) composed of a synthetic binding unit and a synthetic address unit.From this basic idea for a nucleic acid immobilization system, manyvariations of immobilized nucleic acid arrays may be realized. Theseinclude both passively and actively assembled arrays of immobilizednucleic acids which differ in terms of sequence, origin (e.g., fromdifferent samples), type (RNA, DNA, etc.), or in other ways. Byutilizing the disclosed synthetic binding systems, relatively complexmixtures of nucleic acids can efficiently and specifically be sorted topredetermined locations on a support for facile analysis or further usein nucleic acid based biological assays.

Conjugates for Use in the Present Invention

Thus the basis of present invention are conjugates comprising at leastone nucleic acid (NA) and at least one synthetic binding unit (SBU),generally described by the formula (NA)_(n)(SBU)_(m), wherein n>1, andm>1. Thus, the conjugates of the invention, and for use in the methodsof the invention, may vary by the number and arrangement of nucleic acidand synthetic binding unit components, by the choice of their nucleicacid components, and by their choice of their synthetic binding unitcomponents. Thus it is possible, for example, to link nucleic acids,oligonucleotides or polynucleotides, other supramolecular complexes orpolymers to one or more pRNA, pDNA and/or CNA components so that themolecules form a stable unit, a conjugate, under the conditions requiredfor their use. In this connection, the conjugation need not necessarilybe covalent but may also be carried out via supramolecular forces suchas van der Waals interactions, dipole interactions, in particularhydrogen bonds, complex-type bonds or ionic interactions.

The term “nucleic acids”, as used herein, includes nucleic acids,oligonucleotides and polynucleotides and other molecules which arecapable of specifically hybridizing to their complement (or partialcomplement), and which are composed of natural or modified nucleotides.Molecules regarded as nucleic acids, oligonucleotides or polynucleotidesare all oligomers and polymers which occur naturally or else can beprepared synthetically and which have the ability to hybridize witholigomers of naturally occurring nucleic acids. It should be noted that,as used herein, “nucleic acids” may be differentiable by sequence ofbases, by chemical composition (e.g., DNA vs RNA), or simply by naturalor arbitrarily designated origin (e.g., an amplified sequence from twosamples.) For example, NA₁ and NA₂ may be distinct because they are twoaliquots which have been separately dispensed from the same PCRamplification of the same gene from the same sample cells. Usually,however, nucleic acids will be considered distinct based on sequence ororigin from a separate sample.

Nucleic acids have a somewhat chemically repetitive structure made upfrom monomeric recognition nitrogen heterocyclic base units linkedthrough a backbone, usually furanosyl sugar with phosphodiester bridgesin naturally occurring nucleic acids. Nucleic acids, oligonucleotidesand polynucleotides normally have a linear polymeric structure, butbranched nucleic acid structures have been devised using chemicalmodifications and various branching moieties during chemical synthesis.Examples of naturally occurring nucleic acids are DNA and RNA, in whichthe nucleoside monomers comprising 2-deoxy-D-ribose and D-ribose,respectively. In DNA and RNA, the sugars are both in furanose form, arevia phosphodiester bonds to form the backbone of the polymer. TheN-glycosidically linked nitrogen heterocyclic bases form the specificrecognition structure which allows DNA and RNA to specifically pairbased on the sequence of the bases.

Examples of non-natural oligonucleotides and polynucleotides are thechemically modified derivatives of DNA, and RNA such as, for exampletheir phosphorothioates, phosphorodithioates, methylphosphonates,2′-O-methyl RNA, 2′-fluoro RNA. In addition, more structurally differentmolecules which can pair with DNA and RNA are also included within thedefinition of nucleic acids, like locked nucleic acids (LNA) or peptidenucleic acids (PNA) (Sanghivi, Y. S., Cook, D. P., CarbohydrateModification in Antisense Research, American Chemical Society,Washington 1994; Uhlmann; Peyman; Chemical Abstracts 90(4), 543-584(1990)). A key characteristic of all nucleic acids, oligonucleotides andpolynucleotides, as defined herein, is their ability to pair with orbind to the naturally occurring nucleic acids.

A special category of nucleic acids, as defined herein, are aptamers oraptazymes. These molecules, which are composed of nucleotides, which arefunctionally (if not structurally) distinguishable by their specificaffinity, or binding to target molecules. The molecular recognition ofaptamers and aptazymes is based on binding which is mediated via thespatial structure and dynamics and the spatial presentation of parts ofthe molecule, similar to the antibody-antigen recognition principle. Thepolar nitrogen base moieties of the nucleic acid aptamers, arranged bythe sequence of the nucleotides, are thus able to form a spatiallyarranged “hand” to grab the target molecule. In addition to theirspecific recognition properties, aptazymes possess a catalytic function.For example by brings a pair of reactant target molecules into proximityin the proper spatial relationship, the aptazyme can produce a favorablethermodynamic environment to promote the reaction.

Nucleic acids in accordance with this definition also include thosemolecules which contain in addition to the nucleic acid sequencerequired for pairing or molecular recognition of potential targetmolecules further parts which serve other purposes such as, for example,detection, conjugation with other molecular units, spacing or branching.The molecules include in particular the covalent or stable noncovalentconjugates of nucleic acids, oligonucleotides and polynucleotides withfluorescent dyes, peptides, proteins, antibodies, aptamers, organic andinorganic molecules.

In particular, one may desire to label the nucleic acid, or the SBU, ofa conjugate with a detectable labeling moiety. Often, the labelingmoiety is attached to the conjugate at the end of the NA or SBU which isopposite the site of conjugation, as illustrated in FIG. 5. Labels areuseful for several purposes in the systems of the invention, includingensuring that a nucleic acid has been immobilized at a particularlocation by the SBS, and quantifying an enzymatically modified conjugatethat has, e.g., incorporated a labeled base, or had a quencher cleavedoff, as in FIG. 20. Preferred labeling moieties for use in the presentinvention include fluorescent moieties, quencher moieties, visible dyemoieties, radioactive moieties, chemiluminescent moieties, biotinmoieties, hapten moieties, micro-particle (e.g., visible colloidal goldmicrospheres, fluorescent microspheres, etc.) moieties, (para)magneticmicro-particles, and enzymatic labeling moieties. In particular,fluorescent moieties are preferred because of their easy handling andsafety. Suitable fluorescent moieties for use in the present inventioninclude BODIPY™ dyes, cyanine dyes, Alexa™ dyes, fluorescein dyes,rhodamine dyes, phycoerythrin dyes, coumarin dyes, Texas Red dyes,Lissamine™, FAM, HEX, TET, TAMRA, ROX, EDANA,4-Acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid,4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid, Succinimidyl pyrenebutyrate, Acridine isothiocyanate, Cascade Blue, Oregon Green, LuciferYellow vinyl sulfone, and IR1446 (Kodak™ Laser Dye). In addition tofluorescent moieties, one may wish to use a quencher moiety to absorbparticular wavelength of light. Suitable quencher moieties for use inthe present invention include Black Hole Quencher™ moieties, DABCYL,Reactive Red 4 (Cibacron Brilliant Red 3B-A), Malachite Green,4-Dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), and4,4′-Diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid moieites.

The partners of a binding system binding specifically to one another aredenoted by binding unit and address unit. The part of the binding systemwhich is fixed to the support materials is referred to as syntheticaddress unit (SAU), while the nucleic acid-conjugated part contains thecomplementary synthetic binding unit (SBU). Thus, the nucleic acids inthe conjugates used in the present invention are linked to syntheticbinding units which are able to specifically recognize a complementarysynthetic address unit fixed to a support material. Synthetic bindingunits (SBUs) and synthetic address units (SAUs) useful in this inventionare molecular units which are capable of a specific molecular pairing.When the SBU and SAU recognize and pair, the two form a stable andspecific supramolecular complex, or a synthetic binding system (SBS.)Although the SBU/SAUs which may be used in the invention are not limitedto a particular molecular species (e.g., pRNA, pDNA, or CNA), they maybe described in terms of their key functions and characteristics.

A characteristic of the binding systems is that binding of thecomponents of the binding system is normally reversible and that theposition of the equilibrium between free and complexed components of thebinding system is may be influenced by temperature, pH, concentrationand other solution conditions, in order to promote the binding event, orto strip off the associated SBUs from their corresponding SAUs. Anotherprimary characteristics of synthetic binding systems in accordance withthis invention are that they are of non-natural origin, and do not mimicthe spatial relationship of nucleic-acid-hybridizing structures. Thus,the SBU/SAUs used in the present invention have been preparedsynthetically, and have the advantage of not interacting with naturalnucleic acids. Another advantage is the ability to form more stablecomplexes than nucleic acids of natural origin, in particular withrespect to enzymatic degradation processes. Since synthetic bindingsystems do not pair with, or hybridize or bind to nucleic acids,oligonucleotides or polynucleotides, the use of such systems for sortingand immobilizing nucleic acids does not lead to unwanted interaction orinterference by the immobilization component with the nucleic acids(NA), other biomolecules or other components of the sample.

Preferred synthetic binding systems are nonhelical pairing systems whichhave an elongate linear polymer structure. In general, such structuresare polymers with monomers with the general formula:

wherein B_(b) is a backbone moiety which connects the monomeric unit tothe oligomer, and wherein R^(s) is a specific recognition moiety whichprovides the molecular interaction which allows the SBU to specificallyinteract with the corresponding synthetic addressing unit. The specificmolecular interaction provided by R^(s) may be through any non-covalentinteraction, such as hydrogen bonding, Van der Walls forces, ionicinteractions, etc. Such polymeric structures have the benefit ofincorporating an informatic component into the SBS, in that manydifferent combinations of a few basic monomer units may be created whichspecifically pair with each other. For instance, if the basic nucleotidebases (A, C, T, G) are used as the R^(s) moieties, 4⁴=256 combinationsof a four monomer sequence may be made. Although nitrogen containingheterocycles which specifically interact through hydrogen bonding arepreferred R^(s)'s for use in the invention, other specific recognitionmoieties could be devised by one of skill in the art. In general,preferred nitrogen containing heterocycles are the naturally occurringpurine and pyrimidine bases, as they may be readily obtained in quantityfrom natural nucleic acid sources. However, the use of synthetic ormodified nitrogen heterocycles, such as tryptamine, is not uncommon inSBUs or SAUs, and is also contemplated in the invention.

In general, backbone components for use as B_(b) may be any readilylinked monomeric component which is capable of holding the R^(s) inposition for interaction with its complement, but which also ensuresthat the spatial relationship of the R^(s) moieties does not allow themto interact with naturally occurring nucleic acids. Applicants havefound that six-membered ring structures, preferably those containingcarbon, and optionally containing heteroatoms such as O, N, or S, areeffective for this purpose. In particular, pyranosyl sugar ringstructures have been useful, as they allow chemical reactions andprocessing which are largely analogous to traditional nucleic acidchemistries. However, cyclohexyl rings have also been employed asbackbone moieties. Also, other types of molecules, such as crown ethers,azo-crown ethers, polycyclic carbon structures, or other molecularstructures could be used.

Particularly preferred monomeric units which can be utilized for thesynthesis of synthetic binding systems in accordance with this inventionare, for example, pRNAs, pDNAs or CNAs. Such building blocks aredescribed, for example, in WO 98/25943, Helv. Chim. Acta 1993, 76,2161-2183, Helv. Chim. Acta 1995, 78, 1621-1635, Helv. Chim. Acta 1996,79, 2316-2345, Helv. Chim. Acta, 1997, 80, 1901-1951, WO 99/15539(“pRNA”), WO 99/15509 (“CNA”) and by Beier, M.; et al.; Science 283,699-703 (1999).The useful characteristics of these molecules are alsogenerally applicable to largely analogous structures having, e.g., adifferent linkage moiety besides phosphate between the sugar residues ofthe backbone, or other minor alterations. It is interesting to note thatSBUs and SAUs using these types of monomeric units may be made up ofdifferent classes of monomers, so long as the specific recognition ismaintained. Heterologous binding systems which are preferred here arepRNA/pDNA, pRNA/CNA or pDNA/CNA. However, it is still more preferredthat the SBU and SAU utilize the same class of monomers. pRNA, pDNA orCNA form synthetic binding systems (SBS) which pair in a selective andstable way. The stability of pair formation depends on the number,composition and sequence of monomeric building blocks in the oligomericbinding system and on the number of base stacking interactions betweenthe strands (inter-strand base stacking). Unlike naturally occurringnucleic acids, the strength of the SBS interaction for pRNA, pDNA, andCNA is initially stronger than for a nucleic acid utilizing the samesequence, and the strength of the interaction increases over a largerdynamic range the length of the paring sequence increases. Thus, SBSsmay be easily devised with pRNA, pDNA, and CNA which have an increasedbinding strength greater than that of natural nucleic acids, and thatwill remain bound under conditions which are stringent for the nucleicacid sequence portions of the conjugates. The SBU region or the SAUregion producing a specific recognition preferably comprises from 3 to15, more preferably from 6 to 10, monomers. Specifically usefulsequences for use in libraries or sets of conjugates are describedbelow.

The synthetic binding systems (SBS) or components thereof (SBU and SAU)on their own cannot be synthesized, amplified, modified, processed,ligated, fragmented or hydrolyzed by enzymes which are known fromnucleic acid technology, such as polymerases, ligases, nucleases,restriction enzymes, etc., for example. This property is particularlyadvantageous when using conjugates of synthetic binding units andnucleic acids, since the part of the associate, which relates to thesynthetic binding system, is not modified, blocked, removed or processedby the enzymatic steps normally necessary for processing the sample orthe nucleic acid, such as a preceding amplification, for example. Theproperty is also advantageous, because the synthetic binding system(SBS) or its components (SAU/SBU) are not subject to degradation byenzymes which may be contained in the sample.

As mentioned above, the synthetic binding systems (SBS) and/or thesynthetic binding units (SBU) and/or the synthetic address units (SAU)may contain additional molecular groups which are used for detection,conjugation with other molecular units, spacing or branching. Themolecular groups include in particular the covalent or stablenoncovalent conjugates of pRNA, pDNA or CNA with labeling moieties asdescribed above.

Specific examples of such conjugates utilizing pRNA, pDNA, and CNA forthe SBU component have been described previously in WO99/15893 (U.S.Ser. No. 09/509,051, filed Mar. 20, 2000), WO99/15542 (U.S. Ser. No.09/509,011, filed Mar. 20, 2000), WO99/15541 (U.S. Ser. No. 09/509,039,filed Mar. 20, 2000), and WO99/15509 (U.S. Ser. No. 09/509,040, filedMar. 20, 2000), incorporated herein by reference in their entirety. Inaddition to the newly developed conjugates described herein, these arealso useful in the novel array construction methods, supermolecularstructures, biological assays, and enzyme modification methods of theinvention.

Particular Conjugates Structures for Use, and Their Production

Where both n and m are 1, the conjugate comprises a single SBUconjugated to a single NA. These are the most basic types of conjugatesfor use in the invention. These conjugates have the general formula:(NA)-X—(SBU)in which

-   -   (NA) is a nucleic acid, preferably a DNA, RNA, modified DNA, or        modified RNA, as further described below. The nucleic acid is        preferably linked covalently to X via the 3′ or 5′ oxygen of one        of the terminal nucleotides. See FIG. 1    -   (SBU) is a synthetic binding unit as described above, preferably        a pRNA, pDNA or CNA or a modified form thereof. Preferably, and        the synthetic binding unit is linked to X preferably via a 2′ or        4′ carbon (or the equivalent carbon of the cyclohexyl CAN        moiety) of one of the terminal monomers. However, linkage        through a nitrogen heterocyle moietiy, such as through a        tryptamine linker, is also within the present invention. See        FIG. 1.    -   X is a linking group suitable for conjugating the nucleic acid        with the synthetic binding unit. Although several such groups        could be devised by one of ordinary skill in the art; preference        is given to linker groups having the one of the following        structures (oriented in either direction):        and where    -   Y₁,Y₂ are independently of one another OH; SH, NH₂ or CH₃ and        where    -   G is O or S and where    -   L₁, L₂ are, independently of one another, linkers selected from        the group consisting of:        -   a covalent bond; and a linker chain moiety comprising a            saturated or unsaturated, branched or unbranched,            substituted or unsubstituted, chain of 1-60 carbon atoms and            0-40 heteroatoms selected from the group consisting of N, O,            and S. Exemplary non-covalent bond linkers preferred linkers            include            —[—(CH₂)_(n)—]—            or —[—CH₂—CH₂—(O—CH₂—CH₂)_(]—)            or —[—CH₂—CH₂—CH₂—(O—CH₂CH₂—CH₂)_(q)]—            or —[—(CH₂)_(v)—C(O)NH—(CH₂)_(z)—]—            or —[—(CH₂)_(v)—NHC(O)—(CH₂)_(z)—]—            with n, m, q, v, z in each case independently of one another            being an integer between 1 and 20, and more preferably, n is            between 2 and 12 , m is between 1 and 5, q is between 1 and            4, and v and z are independently between 2 and 6; and where    -   V₁, V₂ are independently of one another a group selected from        —[—CH₂—]—        and        and where    -   R₁, R₂ are independently of one another H or OH, and where    -   Ba is a nitrogen heterocycle moiety, such as, e.g., an indole        linker or a base of the kind that is normally utilized in        naturally occurring or man-made nucleic acids (adenosine,        cytosine, guanine, thymidine, idenosine, etc.) and synthetic        binding systems.

Conjugates using X structures:

are particularly preferred for use in the invention, as pRNA and pDNAembodiments may be readily synthesized utilizing solid phasephosphoramidite synthesis methods. To make the novel L linker conjugatesof the invention, a number of “spacer” phosphoramidite reagents with awide variety of L moieties care available from commercial sources suchas Glen Research (Sterling, Va.). These include polyethylene glycolchains, alkyl chains, a-basic ribose, etc. In addition, the organicchemist of ordinary skill may synthesize suitable linkerphosphoramidites from suitable polyether, polyester, polyamide, or otherspacer moieties. Various linkage arrangements are illustrated in FIG. 2.

An option for preparing conjugates with a free 3′ end in the nucleicacid portion is to start the synthesis of the binding system on the CPGsupport, and then assemble the nucleic acid after the SBU. If reversebuilding blocks are used for this purpose, it is possible to synthesizethe nucleic acid in the 5′→3′ direction. The result is a linkage asdepicted in FIG. 1B and FIG. 2B/2E. As long as no free 3′ end isrequired in the nucleic acid part, the nucleic acid synthesis may becarried out with standard building blocks on the binding unit. Theresult is a linkage as depicted in FIG. 1A and in FIG. 2A/D (cf.:DE19741715 or WO99/15539, “Pentopyranosylnucleosid, seine Herstellungund Verwendung”).

Applicants have recently devised improved methods for the direct solidphase synthesis of pRNA, pDNA, or other phosphate-backbone conjugates.It was surprising for applicants to find that it is possible to prepareconjugates of synthetic binding systems and nucleic acids particularlyeasily in a chemical solid phase synthesis, if a particular protectivegroup strategy is applied. The allyl protective group used previously,and its deprotection on the pRNA phosphoester using palladium catalysts,led to partial fragmentation of the synthesized oligomer duringdeprotection when relatively long strands were synthesized on thesupport. In contrast, using a β-cyanoethyl protective group and thestandard hydrazine deprotection step leads to very low yields of thedesired conjugate pRNA. The removal of the β-cyanoethyl group byhydrazine, and desired conversion into a phosphodiester which can bedeprotected by the hydrazine, is slower than the undesired side reactionof nucleophilic cleavage of the phosphotriester bonds of the conjugateby hydrazine. Surprisingly, treatment of the pRNA-DNA conjugate whilebound to the CPG support with a solution of an alkylamine in an inertsolvent, after the completion of synthesis, avoids this problem. Aftertreatment, the β-cyanoethyl protective group may be deprotected withhydrazine without cleaving the pRNA-DNA conjugate. Preferably, asecondary alkylamine in an inert solvent is used. Preferred secondaryalkylamines include dimethylamine, diethylamine, dipropylamine,dibutylamine, dipentylamine, dihexylamine, di-N-octylamine,di-N-decylamine, didodecylamine, N-ethylmethylamine,N-methyl-N-propylamine, N-methylbutylamine, N-methylpentylamine,N-methylhexylamine, N-ethylpropylamine, N-(N-butyl)-N-propylamine,N-amyl-N-butylamine, N,N′-di-N-butyl-1,6-hexanediamine,N,N′-dimethyl-1,3-propanediamine, N,N′-dimethyl-1,6-hexanediamine.Diethylamine is particularly preferred. The alkylamine is preferably insolution at a concentration of about 0.2% to about 10%, more preferablyfrom about 1% to about 5%, and more preferably at a concentration ofabout 1.5%. Suitable solvents for use include dichloromethane,chloroform, carbon tetrachloride, dichlorethane, tetrahydrofuran,toluol, diethylether, ethanol, methanol, acetonitrile, hexane, andheptane. Applicants have found an about 1.5% solution of diethylamine indichloromethane to be particularly suitable. The intermediate obtainedafter treatment can readily be deprotected by hydrazine and purifiedchromatographically. Thus, directly synthesized pRNA-DNA conjugates maynow be made easily in good yield.

It is often desirable to link pre-synthesized binding systems tofinished nucleic acids. Nucleic acids which are obtained from naturalsources or via enzymatic reactions such as, e.g., in vitro transcription(also frequently used for preparing aptamers and aptazymes) are moreadvantageously conjugates to SBSs with a functional linker. However,many biochemical conjugation methods described in the literature (e.g.Hermanson, G. T.; Bioconjugate Techniques, Academic Press, San Diego1996) have proved chemically unsuitable for the nucleic acids and/or thesynthetic binding units. Several methods for linking nucleic acids withnucleic acids utilize the hybridization of the nucleic acids in order toachieve initial attachment and positioning, followed by covalentlylinking the strands (e.g., photochemically via psoralen.) Such methodare not useful for SBU/NA conjugation, as the SBUs do not pair withnucleic acids.

Applicants disclose several novel conjugates based on hydrazine andhydrazide chemistries for linking pre-made SBUs and NAs together intoconjugates. Hydrazide-modified nucleic acids or synthetic binding unitshave advantages compared with amino-modified nucleic acids or syntheticbinding units, because they can be used over a wider pH range, andbecause the products formed are relatively stable, even without thereduction step required for the amino-modified nucleic acids orsynthetic binding units. Hydrazine conjugation chemistries have beenutilized for nucleic acids, as described in WO 00/19653. However,applicants have found that hydrazine chemistries are particularly usefuland effective for coupling pRNA, pDNA, and CNA SBUs to nucleic acids.For instance, conjugates with X of the formula:

may be obtained by reacting a first conjugate moiety (the SBU or NA ofthe conjugate) containing a hydrazide with a second conjugate moietycontaining an terminal ribonucleoside (or simply a ribose, without abase). This is illustrated in FIG. 5. An advantage of this method isthat the nucleic acid to be conjugated does not have to be chemicallymodified: either a naturally occurring terminal riboside may be used, orone may be added to a deoxyribonucleic acid enzymatically with aterminal transferase. The ribonucleotide may be oxidized with periodateto give the corresponding aldehydes, which then react with amines orhydrazides to give stable conjugates.

In addition, applicant has developed several other novel hydrazide andhydrazine coupled conjugates for use in the invention. Those with theformula:

can be produced by reacting an aldehyde first moiety with a hydrazide orhydrazine second moiety, respectively. Example 2.2 shows a method forpreparing a hydrazide-modified pRNA conjugate. An aldehyde function isobtained at the 3′ end of the oligonucleotide, if the nucleic acid issynthesized on a commercially available glyceryl support (e.g.: GlenResearch Corp., Sterling, Va., USA; catalog No. 20-2933-41) andsubsequently oxidized with sodium periodate. This variation isparticularly useful conjugation with RNA, since the first RNA buildingblock at the 3′ end is no longer attacked by periodate, remainingcompletely intact. If it is desired to introduce the aldehyde functionat the 5′ end, it is possible during synthesis to link up aphosphoramidite building block which either already contains a protectedaldehyde function or includes a protected diol (e.g. described in EP 0360 940), which can be cleaved subsequently by means of periodate. Thetwo components are reacted in phosphate buffer, pH 7.4, in the presenceof sodium cyanoborohydride, and the product is isolated (e.g., byRP-HPLC).

In addition, the novel conjugates linked by an X of the formula:

are useful in the invention. These may be obtained by a method analogousto that of the hydrazide conjugates by reacting a semicarbazone orthiosemicarbazone containing first conjugate moiety with an aldehydecontaining second conjugate moiety.

An additional conjugate for use in the present invention comprises and Xof the formula:

which may be obtained by treating a thiol on a first conjugate moietywith iodoacetate, and then reacting it with an amine on a secondconjugate moieity. See

In addition to the relatively simple conjugates described above, morecomplex novel conjugate structures wherein n, m, or both, are ≧2 may beeasily prepared using the guidance provided herein. Where n≧2, theconjugate comprises multiple nucleic acids. Likewise, when m>1, theconjugate comprises multiple SBUs. Using a method for preparing andpurifying conjugates of synthetic binding units and nucleic acids it ispossible for various conjugates to be prepared and tested for theirapplicability for addressing, sorting and immobilizing nucleic acids(NA) on the support material. Thus, conjugates may be designed whichdiffer by:

-   -   a) the choice of binding unit,    -   b) the choice of nucleic acid,    -   c) the structure of the linkage between nucleic acid and binding        unit,    -   d) the orientation of the nucleic acid,    -   e) the number of synthetic binding units conjugated with a        nucleic acid.

Such multi-component conjugates are may be formed as linear-linked chainstructures, or as branched structures utilizing a branching moiety, asdescribed below. Multi-SBU structures have several uses, including:increasing the immobilization bond strength through the formation ofmultiple SBSs to immobilize one or more conjugated nucleic acids,providing multiple SBUs for use with different arrays of immobilizedSAUs, and providing an extra informational dimensionality to themolecule by choosing conditions under which the SBUs bind to the SAUs torequire multiple SBU/SAU binding events for immobilization (e.g., thenucleic acid is immobilized only at a location to which both SAU₁ andSAU₂ are attached, but not one to which just SAU₁ is attached.) Thislatter alternative is particularly attractive in mass-produced systems,as it allows shorter sequences of synthetic binding system components tobe utilized to convey the same level of specificity as longer sequences(e.g., two 6-mers rather than a single 12-mer.) In addition, byutilizing branching groups or linear structures one may conjugatemultiple NAs to one or more SBUs. This allows the creation of animmobilizable probe structure with multiple functionalities (e.g.,capture probes for two genetic loci, or forward and reverse SDA probesas illustrated in FIG. 25).

The SBUs and NAs of the complex conjugates may be linked together via alinker group X or branching group W, in various configurations. Forinstance, conjugates may be formed with any of the following structures:(NA)-X₁—(SBU)—X₂—(NA)(SBU)—X₁—(NA)-X₂—(SBU)(NA)-W—(—(SBU))_(u)(SBU)—W—(—(NA))_(u)((NA)-)_(u)-W—(—(SBU))_(u)with u being an integer between 2 and 6, preferably 2 or 3, in which

-   -   (NA) Is as defined above,    -   (SBU) Is as defined above,    -   W is a branching group which makes it possible to conjugate a        plurality of synthetic binding systems with at least one nucleic        acid. W preferably has the general formula:    -   wherein Y₁ is OH, SH, NH₂ or CH₃;    -   wherein D₁, D₂, D₃, and D₄, are, independently, a covalent bond        or a linker chain moiety comprising a saturated or unsaturated,        branched or unbranched, substituted or unsubstituted, chain of 1        to 10 carbon atoms and 0 to 4 heteroatoms selected from the        group consisting of O, S, and N;    -   wherein J is carbon or nitrogen;    -   wherein z is 0 or 1, further wherein z is 0 if J is nitrogen;        and    -   wherein X₁, X₂, and X₃, are independently X as described above.

Preferred branching groups include:

When utilizing polymeric SBUs, one of the choices in the construction ofconjugates is the sequence of monomeric units in the SBU and itscorresponding SAU. For use in methods of the present invention wheresets of SBUs and SAUs are utilized to create arrays and othersupermolecular structures, sets of suitable synthetic binding units(SBU) and complementary address units (SAU) are greatly advantageous.Using the described synthetic binding units and address units, it ispossible to define a set of pairing systems which are similar in theirproperties but orthogonal to one another. The term “orthogonal,” as usedherein, means that the members of a set or group of SBUs hybridize withthe other members of the set and the complementary SAU's of the othermembers of the set to an lesser extent than the average hybridization ofall possible sequences of the same length. The concept of orthogonalityis illustrated in the data in FIG. 14. As shown here, most of thesequences in this group are substantially orthogonal to each other. The118 and 119 pairs are less orthogonal to each other than the others,though, and probably should not be utilized in the same set.

In general, set of synthetic binding units and address units preferablycontains a set of different sequences of monomeric units (R^(s)'s)having from 3 to 15, particularly preferably from 6 to 10 monomerbuilding blocks. The R^(s) sequences are preferably orthogonal withinthe group. Nitrogen heterocycle moieties, specifically nitrogenous basemoieties, are very useful because a large number of possiblecombinations of subunits can be made, allowing for the production ofrelatively large, orthogonal groups of SBUs. The described SBUs and SAUsshow mutually orthogonal behavior under high stringency conditions, andin this respect are substantially more efficient than DNA or RNA. It is,however, in addition possible to define individual subgroups of the set,which show mutually orthogonal behavior even with less stringentconditions.

In particular, the applicants have found useful sets of syntheticbinding units and synthetic address unit, having the structure:

-   -   wherein B_(b) is a backbone moiety which connects the monomeric        unit to the oligomer (e.g., a pyranosyl or cyclohexyl ring),        wherein R^(s) is a specific recognition moiety comprising a        nitrogen heterocycle moiety.    -   wherein the monomeric units are linearly arranged according to        the formula,        B_(s1)-(J)-B_(s2)or        B_(s1)-(J)-B_(s2)-(J′)-B_(s3)

wherein s1, s2 and s3 are, independently, an integer between 0 and 10,more preferably between 0 and 2, and B is any monomeric building blockas is used for synthesizing the synthetic binding units (SBU), and J isa sequence of recognition moieties (R^(s)), wherein J may be the same ordifferent than J′. In these structures, the sequences J and J′ arepreferably, independently, selected from group A described below, or areselected from the group B, described below, and sequences J and J′ areselected from the same group SEQ. ID SBU/SAU pRNA Sequence, No. Ref. No.4′→2′  1  1a AATGCCTA  2  1b TAGGCATT  3  3a AATCGCTA  4  3b TAGCGATT  5 4a AAGTCCTA  6  4b TAGGACTT  7  6a AATGTCCA  8  6b TGGACATT  9  7aAATCCGTA 10  7b TACGGATT 11  10a AATTCGCA 12  10b TGCGAATT 13  11aAACGTTCA 14  11b TGAACGTT 15  12a AGTACTCA 16  12b TGAGTACT 17  13aAATCTCGA 18  13b TCGAGATT 19  14a AAGCTCTA 20  14b TAGAGCTT 21  15aACTAGCTA 22  15b TAGCTAGT 23  16a AAGTTCCA 24  16b TGGAACTT 25  17aAAGCCTTA 26  17b TAAGGCTT 27  18a ATGACCTA 28  18b TAGGTCAT 29  24aAACGCTTA 30  24b TAAGCGTT 31  25a ACTGACTA 32  25b TAGTCAGT 33  28aAATTGCCA 34  28b TGGCAATT 35  29a AACCGTTA 36  29b TAACGGTT 37  30aAGTCACTA 38  30b TAGTGACT 39  31a AACTGCTA 40  31b TAGCAGTT 41  34aCTGGCATA 42  34b TATGCCAG 43  35a CCAGTCTA 44  35b TAGACTGG 45  36aAATGCGTA 46  36b TACGCATT 47  37a AATCCTAG 48  37b CTAGGATT 49 102aTCCTGCATTC 50 102b GAATGCAGGA 51 103a CTCTACGTCT 52 103b AGACGTAGAG 53104a CCTCGTACTT 54 104b AAGTACGAGG 55 105a TTCTGTATCC 56 105b GGATACAGAA57 106a CTTTATGCCT 58 106b AGGCATAAAG 59 109a CCCACTTGTT 60 109bAACAAGTGGG 61 110a TCTGCTCATC 62 110b GATGAGCAGA 63 113a CTCACCTATT 64113b AATAGGTGAG 65 117a TTCTATACTC 66 117b GAGTATAGAA 67 118a TGTTTGGGTG68 118b CACCCAAACA 69 119a TGGTCGGTTG 70 119b CAACCGACCA 71 120aTGGTTATCTG 72 120b CAGATAACCA 73 121a CGTGTATGTA 74 121b TACATACACG 75122a CTCCATGTTC 76 122b GAACATGGAG

Thus, substantially orthogonal groups of SBUs or SAUs may be produced bysimply selecting any number (2, 5, 10, etc.) of sequences for use asSBUs or SAUs in the set from either group of sequences below:

-   -   Group A: SEQ. ID Nos. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,        25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,        57, 59, 61, 63, 65, 67, 69, 71, 73, and 75    -   Group B: SEQ. ID Nos. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,        26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,        58, 60, 62, 64, 66, 68, 70, 72, 74, and 76

More preferred subgroups of sequences from which to choose syntheticbinding units and synthetic addressing units for use in arrays requiringstrict orthogonality for sorting are given below:

-   -   Group A′ (8-mers): SEQ. ID Nos. 3, 5, 7, 9, 11, 13, 15, 17, 19,        21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47    -   Group B′ (8-mers): SEQ. ID Nos. 4, 6, 8, 10, 12, 14, 16, 18, 20,        22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 48    -   Group A″ (10-mers): SEQ. ID Nos. 49, 51, 53, 55, 57, 59, 61, 63,        65, 67, 69, 71, 73, and 75    -   Group B″ (10-mers): SEQ. ID Nos. 50, 52, 54, 56, 58, 60, 62, 64,        66, 68, 70, 72, 74, and 76    -   Group A′″ (more preferred 10-mers): SEQ. ID Nos. 49, 51, 53, 57,        59, 61, 65, 69, 71, and 75    -   Group B′″ (more preferred 10-mers): SEQ. ID Nos. 50, 52, 54, 58,        60, 62, 66, 70, 72, and 76

For use in sets of conjugates or addresses, the sets of orthogonalsequences may be present, for example, as a pRNA, a pDNA, a mixture ofpRNA and pDNA or as a CNA. As interactions with native nucleic acids arenot of concern when using the synthetic binding systems of theinvention, the sequences for use in sets of conjugates or addressingunits may be fully optimized for orthoganality, without the requirementfor deleting any naturally occurring sequences. This is a considerableadvantage over the use of nucleic acids as immobilization moieties inprevious methods.

SAUs for Use with the SBU Conjugates in the Invention

SAUs are the coordinating specific binding pairs of the SBUs of theconjugates, and so generally share similar characteristics with theSBUs. The main difference is that SAUs are attached to a supportmaterial, rather than being conjugated to a nucleic acid or otherbiomolecule. The SAUs may be attached to a single solid support (as in aplanar rectangular array) or to several distinct supports (as in a setof beads, dipsticks, or the wells of a microtiter plate). Preference isgiven for utilizing sets of binding addresses which do not cross-reactunder the particular application conditions (e.g., physically separatedin a planar array, vs. able to interact with each other on a set ofbeads) and which react in a similar way to stringency conditions suchas, for example, pH, ionic strength, temperature, electrical potential,denaturing agents, etc., and which show similar stability properties.Thus, the SAUs preferably belong to one molecular class (e.g., all pRNA,all CNA, etc.). For specific embodiments, however, it is also possibleto combine SAUs with different properties on the same support.

Any suitable support material may be used as the surface to which theSAU is attached. For instance, solid materials insoluble under theapplication conditions may be used. These include silicon, silicondioxide, silicon nitride, controlled porosity glass, ceramics, metals,preferably precious metals, in particular gold or platinum, orsemiconductors, metal silicilide, plastics and polymers, includingconductive polymers. In addition porous materials such as, inorganicsol-gels, and hydrogels or biopolymers may be used as support materials.These include, for example, cellulose, agarose polyacrylamide,polymethacrylamide, and organic polymer hydrogels. The surfaces of thesupport materials can be a single material, or several materials inlayers or coatings. The coatings may be in the form of monomolecularlayers, of a coating containing a plurality of stacked layers, or of acoating containing disordered layers. The structure of the support layerwill depend on its purpose: for instance, an agarose bead or siliconwafer surface may simply serve as a solid support to localize the SAU ina particular known position. Sensor arrays, such as the SPR devicesutilized in the experiments, the support may comprise a derivatizedmetal surface for the direct attachment of the SAU to the sensor.Conversely, active electronic arrays, as utilize in the activesupramolecular array construction below, comprise an electrode at thearray locations below a permeation layer (e.g., agarose orpolyacrylamide hydrogel).

As the SBSs of the invention may be utilized particularly advantageouslywith active electronic arrays (as the devices are described in the abovereferenced patents, incorporated herein by reference), this particularembodiment will be described in more detail. A particular characteristicof permeation layers utilized in active electronic arrays is that theyare porous, allowing penetration of conjugates, nucleic acids or samplesdeep into the layer, and providing a large surface area for interactionsto occur. These permeation layers also separate the nucleic acids at theactivated locations from the harsh electrochemical environmentimmediately at the electrode. Furthermore, the surfaces of supportmaterials or of polymers applied to support materials frequently havefunctional groups which allow the covalent or stable non-covalentattachment or linkage of address units of the binding systems. Examplesof such functional groups are activated esters, aldehydes, thiols,amines, as well as streptavidin, avidin, and other biotin-bindingproteins. etc.

In addition to the active electronic arrays (as described in the abovereferenced patents, and also in U.S. Pat. No. 5,605,662, incorporatedfully herein by reference), typical support materials also include otherdevices, such as sensor chips, chips with flow-through systems orchannels, fluidic systems, beads, magnetic beads, microtiter plates,stationary chromatographic phases and also other solid materials whichcan be used in heterogeneous assay systems.

Passive and Active Methods of Producing Supramolecular Array Structures

As described above, conjugate formation makes it possible to encode theindividual nucleic acids (NAs) with one or more synthetic binding units.Encoding is defined herein as the unambiguous assignment of aspecifically addressable synthetic binding unit to a nucleic acid sothat subsequently the nucleic acid is specifically, able to be spatiallylocated, immobilized, sorted, and thus detected on a support materialvia the specific interaction between address unit and complementarybinding unit. Thus, sets or libraries of conjugates can be prepared inwhich various NAs are encoded by one or a group of SBUs. For example,one can produce a library in which all NAs are encoded by a differentSBU, or where all NAs are encoded by a known group of SBUs. Conversely,one can produce libraries in which all SBUs encode a single NA, or aknown group of NAs. If one produces a library in which all NAs areencoded by a single unique SBU, a strict 1:1 conjugate library results.

When contacted with the coordinating SAU under the appropriateconditions for binding, the SBU of a conjugate binds with the SAU toform an SBS-immobilized nucleic acid. The specific binding of thesynthetic binding unit (SBU) to the synthetic address unit (SAU)preferably forms position-resolved synthetic binding systems (SBS) andconstructs, and this leads to the sorting and/or immobilization of theconjugates onto the surface of the support at particular locations wherethe SAUs are attached. Thus, it is advantageous to attach SAUs, ordefined groups of SAUs, to predetermined locations on the support, sothat the identity of the immobilized nucleic acids may be determined ina position-dependent manner. For instance, the SAUs may be attached inan array of locations (rectangular, circular, or any other suitablepatern), in order to produce an array of immobilized nucleic acids atthe predetermined locations. An array of this kind is preparedanalogously to the methods used in DNA chip technology (cf.: MicroarrayBiochip Technology; Schena, M. Eaton Publishing, Natick, 2000; DNAMicroarrays, Schena, M., in Practical Approach Series, Oxford UniversityPress, Oxford 1999). The attachment of the synthetic address units onthe support material via a covalent linkage is particularlyadvantageous, although biotin/streptavidin type systems have also beenused with good results. Branched systems in which one SAU is providedwith a plurality of hydrazides as reactive groups have proved useful, inparticular with active ester permeation layers on active electronicarrays. The provision of a multiplicity of reactive groups in a moleculeleads to a faster and more stable immobilization on the support material(PCT/US00/22205). (See FIG. 4B) If a location comprises a mixture ofSAUs, then several nucleic acids with different SBUs may be immobilizedat a the location (see FIG. 10), or nucleic acids conjugates to multipledifferent SBUs may be immobilized at the location (see FIG. 7).

Thus, in one group of embodiments of the present invention conjugates inwhich a nucleic acid is conjugated with a plurality of synthetic bindingunits (either the same, or different) are used. Systems of this kind arepossible by using branching building blocks (Shchepinov, M. S.; Udalova,I. A.; Bridgman, A. J.; Southern, E. M; Nucleic Acids Res.; 25, 44474454(1997)) in combination with the above-described methods. See FIG. 7. Theadvantage of such a molecular architecture is that, due to the multiplebinding systems in one conjugate, the interaction of the mobile bindingsystem with the part of the binding system, which is immobilized on thesupport material, becomes multiple and cooperative. As a result, it ispossible to achieve an extremely stable immobilization of the conjugateson the support material for specific applications, while maintaining theselectivity of the binding system. As discussed earlier, these systemsalso allow the use of shorter SBUs to produce a greater variety ofspecific binding pairs than is possible using a single SBU of the samelength. Systems of this kind are novel and the applicants were able toobtain them for the first time.

FIG. 1 depicts some of the possible orientations of conjugates bound toSAUs on the surface of support materials. Depending on application,particular embodiments are preferred. For enzymatic processing of anucleic acid part of a conjugate as a primer for a polymerase reaction,the 3′ end of the nucleic acid is preferably free. This can be achieved,for example, by initiating the chemical synthesis on a CPG support withthe nucleic acid part and then synthesizing the binding unit onto thenucleic acid (FIG. 1C; FIG. 2C) (see: DE19741715 or WO99/15539,“Pentopyranosylnucleosid, seine Herstellung und Verwendung”[“Pentopyranosyl nucleoside, and the preparation and use thereof”]). Itis possible here, where appropriate, as depicted in FIG. 2F to insertsegments as linker or spacer by modifying the nucleic acid or usingcommercially available modified building blocks, as described above.Such a separation of binding unit and nucleic acid is useful whenever,under the application conditions, the spatial proximity of the systemsleads to steric problems, for example to poorer hybridization of thenucleic acid with the sample. Such conjugates may be obtainedparticularly effectively by using the novel deprotection method.

The synthetic binding systems, in particular pRNA, pDNA and CNA, formsubstantially more stable pairs than naturally occurring nucleic acids,such as DNA. Surprisingly, it was found that pRNA oligonucleotidescomposed of eight monomer units (8mers) achieve a stability which ismatched for DNA only by 25mers. Furthermore, the stability of pRNA isdetermined by the number of base stacking interactions between thestrands rather than by the CG content. A strong base-stackinginteraction of this type between the strands via the π electron systemsof the bases is not possible in the helical structure of DNA and RNA(see FIG. 3), as compared to the normal RNA/DNA base-stacking energyeffects. As a result, the temperature stability increases continuouslywith the sequence length of pRNA, pDNA or CNA duplexes, orheteroduplexes, and this makes it possible for the synthetic bindingsystems to tolerate relatively high preparation and assay temperatureswhich are more stringent for the nucleic acid components of theconjugates.

In further embodiments, it is advantageous to use labeled conjugates ofsynthetic binding units and nucleic acids for monitoring, control andquality assurance of the immobilization on the support materials (seeFIG. 9). Suitable labels are here in particular fluorescent dyes, dyes,radioisotopes and enzymes or micro-particles, as described above. Thesemay be detected by fluorescent, luminescent, visible spectrum absorptionor transmission (calorimetric), scintillation counting, or othersuitable means. In one embodiment, the conjugate of synthetic bindingunit and nucleic acid is provided with a label which is differentiablefrom a second label for detecting the interaction between the nucleicacid and sample components. Preferably, “quality control” labels do notinteract, interfere or overlap with the second label. Detection of thelabeled species on the conjugate or detection of the signal generated bythe labeled species indicates whether the conjugate has been immobilizedas desired. In another embodiment, the address unit of the syntheticbinding systems, which has been immobilized on the support material, maybe labeled with a species 1 which can interact specifically with alabeled species 2 which is connected with the conjugate of syntheticbinding system and nucleic acid. See FIG. 9. Typical examples of suchinteractions are fluorescence quenching using quenchers and fluorescenceresonance energy transfer. Preferred groups for fluorescence quenchingare DABCYL and the Black Hole Quencher™ moieties. Systems of this kindadvantageously indicate by the specific interaction the correctimmobilization of the conjugates. Since in the case of fluorescencequenching correct immobilization leads to a decrease in the fluorescencesignal, conjugates immobilized on the support material are obtained,which can be used to carry out further detection by fluorescence withoutinterference of the fluorescence used for detecting the immobilizationof the conjugates.

Supramolecular constructs of immobilized nucleic acids can thus beproduced by passive binding of the SBU/NA conjugates to one or more SAUsattached to a solid support. On passive, diffusion- orconvection-controlled array supports, each binding address allows onlyimmobilization of a particular conjugate provided with the complementarypart of the binding address. This results from the fact that the arrayscan be exposed in each cycle only to one mixture of conjugates, which isidentical over the entire array. Thus, only one cycle (M=1) is possible,as the entire array is contacted with all conjugates of a set ofconjugates C. In this case therefore, the number N of binding addressesdetermines at the same time the number of possible different immobilizedconjugates of synthetic binding units and nucleic acids, which can beimmobilized and sorted on the support material (See FIG. 13).

The specific recognition of the addresses for self-organization allowsthe described method to immobilize in parallel N conjugates andtherefore N different nucleic acids on support materials in one cycle.Likewise, all positions of such arrays can be contacted under theconditions of the application only with one type of sample at the sametime and under conditions identical for all positions of the array. Anadvantage of the embodiment is that the user need not provide anyparticular apparatus or instruments, in order to generate an array ofnucleic acids. The array of synthetic address units is universal and cantherefore be employed for generating a multiplicity of nucleic acidarrays. The user requires only one set or set of nucleic acidsindividually encoded with SBUs. The nucleic acids organize and separatethemselves on the support material with fixed SAUs by the specificmolecular recognition of SAUs and SBUs and provide a specificimmobilization.

Another advantage, as compared to spotting of individual nucleic acids,is that the user, since he applies sets of encoded nucleic acids to thesupport material, is able to carry out all necessary process steps onthe conjugates as an entire set after conjugation of nucleic acids withSBUs. For example, instead of N individual desalting, purification orconcentration steps, only one of those steps is required for the set.This makes it possible to save material and process steps. Moreover,these error-prone steps are all carried out in one step, so that thereis no deviation from nucleic acid to nucleic acid. This eliminatesvariability caused by individual treatment of the nucleic acids, whichcould distort the quantitative quality of data gathered from an assaycarried out on the array.

In contrast to passive array construction techniques, the generation ofan array on an active location device allows the selective sorting ofmultiple set of conjugates. In general, active location array devicesare devices in which individual locations are activatable to produceconditions favorable for SBS formation specifically at the location.Thus, the “activatable” locations are individually selectable in acontrolled fashion. Such activation may be of a chemical (e.g., remotelycontrolled release of a hybridization promoting agent at the location)or physical nature (e.g., electric or magnetic field, IR [heat] orphotonic energy, mechanical exposure [see, e.g, the device produced byClondiag, Germany, described in WO01/02094] etc.). Of particular use andeffectiveness in the array construction methods of the invention areactive electronic arrays, in which individual locations on the array maybe activated by an associated electrode. For example, see U.S. Pat. No.5,605,662 “Active Programmable Electronic Devices for MolecularBiological Analysis and Diagnostics”. Since active electronic arraysmake it possible to actively control the electrokinetic movement andconcentration, or addressing, of synthetic address units (SAU), ofSBU—NA conjugates, and of samples to particular positions of the array,it is possible to immobilize N×M different conjugates on such activeelectronic arrays with a number N of different binding addresses, byperforming M addressing cycles in succession.

Thus, when generating the array of immobilized synthetic address unitsfor use in the active construction of arrays, several positions of thearray are initially (and, optionally, simultaneously) provided withidentical binding addresses. The SAUs may be spotted for high-throughputmanufacturing, or may be electronically addressed, as depicted in FIG.12A. In electronic addressing methods, in each of N steps at least Midentical synthetic address units (SAU) are immobilized in parallel sothat, in the end, a chip array of N different synthetic address units isobtained, with each SAU present in at least M fold repetition.

M mixtures (sets) of up to N conjugates of nucleic acids (NA) andcomplementary SBUs are then addressed to N array positions. In purearray-construction embodiments where all N conjugates to be immobilized,each individual synthetic binding unit complementary to a specificaddress unit is present as a conjugate with a specific nucleic acid, andthe group (set) of the activated locations contains the individualbinding addresses at any locations where the immobilization of thecorresponding nucleic acid (NA) is desired. In some assay-typeembodiments, the user may not initially know if all N conjugates arepresent (e.g., if certain amplicons from a sample are present.) In theseembodiments, the N conjugates present will form SBSs with the SAUs ofthe activated locations, positioning the nucleic acids from the sample.

Utilizing this system, any number of patterns for immobilization may beobtained. Usually, for immobilization of nucleic acids to form an arrayfor later use, the set of the positions addressed in one cycle willcontain each binding address only once. Addressing of the mixtures isrepeated M times, and washing between the steps guarantees that theunbound conjugates of the first set are completely removed before a newset is applied and addressed. Optionally, the activated locations may beelectronically washed by briefly reversing the bias of the electrode atthe locations. If the active electronic array is part of a more complexsample preparation system with collection electrodes, the washed unboundconjugates may be collected at the counter-charged electrode for furtherdisposal. If desired, SAUs, or more preferably mixtures of all SAUs usedor all possible SAUs, may be attached to the counter-electrode to act asa collection “sink” for all conjugates washed from the locations by theelectronic washing step.

This procedure allows a significant time reduction and simplification ofthe method of immobilization of different nucleic acids on an array. Theadvantages are best exploited if the number N of the addresses and thenumber M of the cycles are the square roots of the number of arraypositions. The optimum for a 100-position array are 10 different bindingaddresses employed in 10 immobilization cycles and 10 addressing cycles.Thus, to load the array with 100 different nucleic acids, 20 (=10+10)process steps are required instead of 100 individual immobilizationsteps for the individual nucleic acids. If each step requires 3 minutesto cycle, an array may be loaded in an hour compared to five.

The advantage for a 100-position array is reduction by a factor of 5 intime spent. If the time required for preparing the array from SAUs isnot included in the calculation, the time is reduced by a factor of 10.Since the array of SAUs is universal, the array can be prepared inadvance. Because of these time savings, mega-arrays of 10,000 differentnucleic acids may be generated in 5 hours (2.5 with a pre-made SAUarray,) and 100,000 in 16 hours (8). For this embodiment it isunimportant whether the synthetic address units are fixed in rows andcolumns, as depicted in FIG. 12, or other suitable formats.

One of ordinary skill will appreciate that novel array structures areproduced by the active array construction methods of the invention, ascompared to those generated utilizing passive array constructiontechniques. In a passive array, the finished product is a set of Nimmobilized nucleic acids, immobilized by N distinct SBSs. In contrast,in an array produced by active construction methods, at least twodifferent nucleic acids are immobilized by the same SBS, at differentlocations. In practice, large groups of 10, 20, 100 or more differentnucleic acids will be immobilized at different locations by the sameSBS.

Use of the Conjugates and Supramolecular Array Structures in BiologicalAssays

The passive and active arrays prepared according to the method can beused in complete analogy to the arrays generated by traditional methods,in addition to having several distinctive uses. The arrays can be used,for example, for SNP or STR (short tandem repeats) assays, as describedby Gilles et al WO00/58522, or by Sosnowski et al. U.S. Pat. No.6,207,373, all of which are incorporated fully herein by reference.

One application of the embodiment is the use of nucleic acids encodedwith synthetic binding units for immobilizing capture stabilizeroligonucleotides for SNP assays (Gilles, P. N. et al., NatureBiotechnology, 17, 365-370 (1999)). In the SNP assay, capture stabilizeroligonucleotides or amplified nucleic acids are immobilized on an array.An array of 100 (W) different capture stabilizer oligonucleotides with10 (N) SBS addresses is prepared, according to the method described inExample 1, by preparing conjugates form SBUs and NA. The capturestabilizer oligonucleotides used may be conjugates with a free 5′ or 3′end of the nucleic acid. The capture stabilizer oligonucleotides arecombined to sets of 10 (N) conjugates, each SBU in the set conjugatingwith a specific nucleic acid and each SBU being present in the set onlyonce. 10 (M) sets are prepared in total. An array having 10 (N)different SAUs, with each SAU being present in 10 (M)-fold repetition,is prepared according to the method described in Example 5. The exampleutilizes an active location array whose positions can be contactedindividually (in M cycles) with nucleic acids, conjugates; and sets,preferably an active electronic array. A first set of capture stabilizeroligonucleotides encoded with SBUs is applied to the array. Thisapplication takes place under stringency conditions which make thespecific and stable binding of SBU to SAU possible. The set is contactedwith a group or set of positions, in which a specific SAU occurs in eachcase once (or in as many times as the immobilization of a specificcapture stabilizer oligonucleotide is desired). For active electronicarrays, the contacting is carried out by active electronic directing ofthe sets to the desired positions. The contacting can take placesequentially or in parallel for all positions of the set. The specificbinding of SAU and SBU leads to the sorting and immobilization of theconjugates on the positions such that on each position one specificcapture stabilizer oligonucleotide is immobilized.

The array is then washed, removing all conjugates of the first setexcept for the specifically immobilized conjugates. Then a second set ofconjugates is applied and the above-described steps are repeated. Inthis way, an array is obtained after 10 (M) cycles, in which the 100 (W)different capture stabilizer oligonucleotides are immobilized, and thearray can then be contacted with the sample nucleic acids to carry out acapture sandwich assay. Electronic or passive hybridization assayformats may be utilized, although electronic assay formats areadvantageously utilized with multiple samples. For instance, if in thecapture immobilization steps, 10 sets of 100 different captures arecreated simultaneously on a 1000 location device, then 10 differentsamples may be electronically hybridized individually to each set ofcaptures, and then analyzed at one time in a final reporter probehybridization step. This is an example of an assay where the array isconstructed before the SBU conjugates of the array are contacted withthe sample.

For immobilizing amplified nucleic acids, SBU-encoded primers are used.Such primers allow the incorporation of SBUs directly into the productsof a sample nucleic acid amplification reaction. After carrying out theamplification, and optional combining of amplification products, theencoded amplified nucleic acids may be easily sorted onto sets ofactivated locations by their SBUs, as described above. In amplification,a single set of encoded primers may be utilized multiple amplification,and the products combined for electronic addressing, or multiple sets ofencoded primers may be used for parallel amplification of nucleic acids.In multiplex reactions, treatment (e.g. desalting, purification,concentration) of the encoded amplified nucleic acids can be carried outtogether, leading to a reduction in the requisite steps and material.The sets are then immobilized on the array, as described above, withindividual amplified nucleic acids again being immobilized on individualpositions. The immobilized amplicons may then be analyzed and detectedby passive or electronic hybridization of reporter/stabilizer probes, orby any other suitable method. This is an example of an assay where thearray is constructed after the SBU conjugates are contacted with asample.

As defined herein, target molecules are all compounds which can interactin a sequence-specific way with nucleic acids. For instance, targetmolecules may be particular nucleic acids; nucleic acid binding peptidesor proteins such as: operator proteins, receptors, antibodies orfunctional parts thereof (e.g, Fv fragments, single-chain Fv fragmentsor Fab fragments,) or enzymes; and also nucleic acid binding cellcomponents (e.g., lipids, glycoproteins, filament components), orviruses and virus components (e.g., capsids, viroids), and theirderivatives. such as, for example, their acetates. Usually, however,“target molecule” refers to nucleic acids in a sample.

Also, a “sample” as used herein is any solution and/or mixture ofcomponents of natural or artificial origin, which is contacted withcomponents of the system described here. Biological samples are oftenpreferred, as they are the usual source of nucleic acids of interest.Biological samples for use in the invention are preferably derived froma sample selected from the group consisting of human materials, animalmaterials, plant materials, fungal materials, cell cultures, viralcultures, food samples, and water samples. In this context “materials”encompasses all bodily fluids, secretions, excretions, soft or hardtissues, and other organism-associated matter which may contain nucleicacids, including nucleic acids from infectious organisms. Samples may besubjected to preparation steps, such as purification of nucleic acids,amplification, or other types of enzymatic processing, before use.

Contact between potential target molecules of the sample and the NAs ofthe conjugates may take place before, after, or occasionally duringimmobilization of the NA/SBU conjugates on the support material by theSAU addresses. The presence and/or the amount of targets in the samplespecifically binding to the conjugates (in the case of hybridizationassays,) and/or to the presence, amount or properties of the nucleicacid (NA) itself (in the case of amplified or enzymatically modifiedconjugates) may then be detected by suitable means. In this context,samples include all molecules, biomolecules, mixtures, and reagentswhich are contacted with the nucleic acids (NA) immobilized on thesupport materials in order to derive information about type and/orpresence or frequency of the target molecules in the sample. The samplesmay thus contain, in particular, labeled and/or unlabeled probes,hybridization probes, enzymes and mixtures for enzymatic reactions, orother reagents for a particular assay format. The sample components maybe processed or labeled to suit the particular assay method. Suitablelabels for use in assays in the invention include those listed above forconjugates and SAUs.

An additional aspect of the invention is the unique re-usability of theSAU-addressed supports. The immobilized nucleic acids of the conjugatesmay be relatively easily removed, in contrast to the traditionalimmobilization systems (e.g., covalent or biotin/streptavidin linkages.)The binding properties of the synthetic binding systems can beinfluenced by setting particular stringency conditions (pH, saltcontent, temperature, voltage, denaturing agents, etc.) so as to removeall conjugates of synthetic binding units and nucleic acids from thesupport material. A preferred method is the addition of a basic solution(NaOH, KOH, NH₄OH, etc.) to lower the pH. Alternative methods includethe use of denaturing agents such as dimethylformamide, formamide,dimethylsulfoxide. If the linkage of the synthetic address unit of thesynthetic binding system to the support material is itself stable towardthe conditions, then an array of immobilized synthetic address units(SAU) is retained after a such a stringency treatment. The array canthen be used again for the immobilization of new conjugates of syntheticbinding units (SBU) and nucleic acids (NA), just like a freshly preparedarray of SAUs. Reusing the support material with the immobilizedsynthetic address units (SAU) saves material (support material andaddress units) and time when preparing the array of immobilized SAUs.The use of synthetic binding systems (SBS) is therefore advantageouscompared with the use of irreversible systems such asbiotin-streptavidin, for example. An example of regenerating a supportmaterial is described in Example 8. These embodiments of the inventionare particularly attractive when arrays comprising intricate functionalelements, such as sensor arrays or active electronic array devices, areused.

As briefly described in the SNP example, the methods of the inventionalso provide ways to use of conjugates of synthetic binding units andnucleic acids to sort and address to support materials, in parallel,nucleic acids which are identical as to sequence or composition, buthave been obtained from different sources. This aspect is particularlyuseful in passive arrays, where samples cannot be sorted on supportssimply by activating particular locations in the array. For thedetermination of gene expression patterns, for example, the differentexpression of identical genes may be compared at different developmentalstages, points in time after a stimulus, or in different populations,all on the same passive array. Advantageously, the informationaldimensionality of the synthetic binding systems (SBS) is utilized inorder to encode each gene studied in each sample with an individualaddress (binding unit, SBU) by utilizing SBU-conjugates as amplificationprimers with a cDNA sample.

After the gene amplicons of the samples of different origin are solabeled, they may be combined and further processed together. Thisavoids variability in treatment of the samples individually, and allowsthe uniform and coordinated addressing of the identical nucleic acidsfrom different sources on the same array under precisely the sameconditions. As a result, a considerably higher accuracy and lowervariation (standard deviation) of the data can be achieved. In contrastto the commonly used encoding by different dyes, encoding by specificsynthetic binding units (SBU) allows a considerably higher number ofdifferent simultaneously usable codes. An overlapping of visiblespectrum signals is also prevented, since the encoding here serves tospatially separate the encoded nucleic acids. In contrast, dyes do notallow a separation of this kind, with the result of a frequent spectraloverlapping of the dyes, which makes signal separation more difficult.

An this type of embodiment of the invention of the embodiment, isdepicted in FIG. 13B. Here, different times, stages or populations ofparticular species of nucleic acids, for example particular genes, on anarray are compared with one another. For this purpose, a standard arrayof attached synthetic address units may be utilized, wherein eachposition has a distinct SAU address. Although each gene in each sampleis given a distinct SBU in this passive example, an alternative would beto use repeated addresses and SBU conjugates, as long as the addressesare handled in separate sets using an active location array. The timesto be compared of one species are directed to the positions either as amixture simultaneously, or sequentially. The specific recognition ofsynthetic address units by the synthetic binding units immobilizes oneach array position only the conjugate which specifically pairs with theSAU at that position.

The target nucleic acids in the samples may be detected and/orquantified by means of labels, as described previously for qualitycontrol labels, either on the targets themselves or on hybridizedprobes. In alternative embodiments, mass spectrometry, such asMALDI-TOF, may also be utilized, with or without mass-labelingtechniques. Various detection schemes have been devised for immobilizednucleic acid array formats, and the person of ordinary skill willreadily appreciate their application to assays utilizing the arrays andmethods of the invention.

Enzymatic Reactions Utilizing SBU—NA Conjugates

As has been previously discussed, the conjugates of the invention may beutilized in polymerase reactions for the enzymatic amplification oftarget nucleic acids in a sample. Surprisingly, applicants have foundthat conjugates comprising at least one synthetic binding unit (SBU) andat least one nucleic acid section (NA) may serve as substrates forenzymatic reactions which utilize nucleic acids as a substrate, and arethus compatible with the commonly used enzymatic processes ofbiotechnological production methods. For use in the enzymatic methods ofthe invention, the synthetic binding units of the conjugates preferablyinclude pRNA, pDNA and/or CNA sections, these sections being able tomediate specific and reversible binding by hybridization. The nucleicacid section (NA) comprises at least one nucleotide, or nucleicacid-compatible nucleotide analog.

Thus, the basic enzymatic method of the invention is simply contacting aconjugate with at least one enzyme which utilizes naturally occurringnucleic acids as a substrate, and further contacting the mixture withother reagents necessary for the action of the enzyme. Then, the mixtureis allowed to incubate under conditions suitable for the enzyme for anamount of time sufficient to effect the enzymatic modification of theconjugate. Both the reagents necessary and the conditions will varyconsiderably from enzyme to enzyme and between process utilized fordifferent effects. For instance, the reagents may include an additionaltemplate nucleic acid or target nucleic acid, such as in a polymerase ortemplate-dependent ligase reaction. Depending on the procedure, furtherauxiliary substances such as, for example, buffers, acids, bases,coenzymes, nuclease inhibitors, etc., may be added. Or the reagentscould include nucleotides for a polymerase or terminal transferasereaction. Likewise, conditions such as temperature vary widely between“thermostable” enzymes, such as those derived from Thermus aquaticus andsimilar species, and heat-labile enzymes derived from, e.g., Escherichiacoli. Applicants have found, however, that the conjugates may be usedsimilarly to regular nucleic acid substrates. The use of the conjugatesin a particular known enzymatic nucleic acid reaction will, therefore,require a minimum of experimentation. In addition, combinations ofenzymes may be utilized in typical processes utilizing concerted enzymeactivities, such as TMA (RNA polymerase, RNAse H and reversetranscriptase), and SDA (restriction endonuclease and polymerase).

Thus, the NA/SBU conjugates described are modified by the particularenzymes, utilizing any requisite template nucleic acids, target nucleicacids and/or nucleoside triphosphates (NTPs), depending on the methodused. The term template nucleic acid means a natural nucleic acid whichhybridizes to the nucleic acid section (NA) of the NA/SBU-conjugatesubstrate and serves as template for the linkage or synthesis of thetarget nucleic acid. The target nucleic acid is understood to be anatural nucleic acid which is enzymatically linked to the nucleic acidsection (NA) of the NA/SBU conjugate, or is enzymatically synthesized,modified or degraded by the reaction after contacting the NA of theconjugate. The nucleic acid section (NA) may be covalently linked to thetarget nucleic acid, or may be hybridized to the nucleic acid section(NA) of an NA/SBU conjugate and enzymatically degraded or modified. Thehybridization may also start enzymatic degradation or enzymaticmodification of the nucleic acid section (NA) itself (e.g., by RNAse Hactivity).

The product of the enzymatic modification of the conjugate is aconjugates whose nucleic acid part has been processed. The final productwill thus depend on the enzymatic process employed. Polymerase reactions(including DNA polymerase, RNA polymerase, and reverse transcriptasereactions) produce amplicons conjugated to SBUs. Ligation reactionsproduce SBU conjugates with additional nucleic acid sequences appended.Nuclease reactions (restriction endonuclease, exonuclease, endonuclease,RNAse, DNAse, etc.) cleave a portion of the nucleic acid section of theconjugate off. Terminal transferase reactions add one or morenucleotides to the end of the nucleic acid of the conjugate (such as forlabeling or homopolymeric tailing.) And myriad other enzymatic reactionsare available to phospohrylate, dephosphorylate, methylate, or otherwisemodify nucleic acids.

Preferred NA/SBU-conjugates for use in the enzymatic methods includethose described above. Depending on application, particular embodimentsare preferred. Thus, for a polymerase or amplification reaction, thenucleic acid section of an NA/SBU conjugate should have a free 3′ end.In contrast, ligases require a free phosphate at the 5′ end of a nucleicacid for linkage to the 3′ end of a nucleic acid. In some embodiments ofthe invention, this is a linkage to the 3′ end of an RNA.

Exemplary enzymes include the polymerases used for amplifying nucleicacids, in particular DNA polymerases such as, for example, Taqpolymerase, Vent exo polymerase, Klenow enzyme, T7 DNA polymerase, PolI, T4 DNA polymerase, PFV1 polymerase, Pwo polymerase or Pfu polymerase,in addition to RNA polymerases such as T7 RNA polymerase, T3 RNApolymerase, SP6 RNA polymerase and reverse transcriptase. Furtherexemplary enzymes include nucleases such as, for example, restrictionendonucleases, exonucleases, RNases such as, for example RNase H orRNase A. These additionally include DNA and RNA ligases such as, forexample, T4 DNA ligase, T4 RNA ligase, kinases, methylases, and terminaltransferases or methyltransferases. Reactions with RNA ligases and DNAligases are very useful for producing lengthened conjugates. Inaddition, nucleases are also useful is enzymatic modification assays,such as the quencher-release type restriction endonuclease assaydepicted in FIG. 20.

In a preferred embodiment, the conjugates are enzymatically modifiedpolymerases with the addition of nucleoside triphosphates to produceamplicons with attached SBUs. For this purpose, the NA/SBU conjugatesare used as primer-substrates in polymerase reactions, with the nucleicacid section of the NA/SBU hybridized with a template nucleic acid. Withthe addition of polymerase and the nucleoside triphosphate monomers(NTPs), the polymerase used the template nucleic acid strand to addcomplementary nucleotides to the 3′ end of the nucleic acid section(NA), thus synthesizing a complementary strand. PCR and similarmethodologies are preferred, and strand displacement amplification(SDA), is also preferred.

For use of NA/SBU conjugates as nucleic acid primers in enzymaticreactions of this kind, the conjugates must be stable under the reactionconditions, in particular to the repeated heating and cooling of PCRreactions. Here, the conjugates of nucleic acid sections (NA) withsynthetic binding units (SBU) comprising pRNA, pDNA and/or CNA provedparticularly suitable. Furthermore, the synthetic binding unit (SBU) ofthe conjugate must be neither an inhibitor of nor a competing substratefor the enzyme. Such a competitive in inhibition of the enzyme isadvantageously not exhibited by the NA/SBU conjugates described.

Anchored strand displacement amplification (SDA) is an efficient methodfor multiplex amplification and discrimination of gene targets, whichcan advantageously utilize the novel mixed-address array structures (seeFIG. 24), and branched conjugate structures (see FIG. 25) of theinvention. Anchored SDA facilitates multiplex amplification because theamplification primer sets (forward and reverse) act as discrete units onat each location on the array, such as on the locations of an activeelectronic microchip. The amplification primer sets can be spatiallyseparated into discrete zones on the chip by attaching the primers tospecific binding units (SBU) that bind exclusively to theircorresponding specific address units (SAU) a the locations. This allowsthe creation of discrete zones of amplification that share only enzymesand reagents, wherein the amplification products are rapidly captured bythe high local concentration of primers. This localization of theprimers and amplicons greatly reduces primer-primer interactions whilemaintaining a completely open architecture that greatly simplifies theamplification process.

FIGS. 27A through 27E illustrate the principles of SBU anchored SDA. Forsimplicity, this description is for a single primer pair used to amplifya single target sequence: however, the method can be extended to anynumber of targets and primers desired. Unique SDA primer-SBU constructs(Primer 1 and Primer 2) are electronically addressed to a specificlocation on the microchip. The target is introduced together with theappropriate dNTP, buffers, salts, restriction enzyme, and polymerase.The chip is heated to dissociate the double-stranded template (or thisstep is carried out before introducing the template), and each target iscopied onto its respective SBU-anchored SDA primer by the action of thepolymerase. Extension of the bumper primer creates a new double strandedtarget leaving the target copy (SI) attached to SBU 1. A second bumperprimer initiates polymerization from the distal end of S1 and creates adouble stranded restriction site proximal to the SBU1 attachment point.The process repeats (nick-strand displacement synthesis cycle) toproduce multiple copies of truncated S1. An analogous process can occurat Primer 2-SBU2. This completes the initiation process (Phase 1). InPhase 2, the multiple copies of S1 generated in Phase 1 are captured byPrimer 2-SBU2 which subsequently may be extended by the polymerase.Inclusion of a second bumper primer and introduction of a secondrestriction site results in strand displacement and amplification of S2,leading to exponential amplification (Phase 3). In the absence of thesecond bumper primer and restriction site, the amplified S1 produced atPrimer 1 is captured at Primer 2 and subsequently can be detected viathe hybridization of an appropriately designed fluorescently (orotherwise) labeled reporter probe. Exponential amplification may beallowed to proceed in order to increase amplification. In this case, anadditional unique capture probe-SBU construct can be used to capture theamplification products and confine them to a discrete location on thechip.

In another preferred embodiment of the invention, NA/SBU conjugates areligated with a target nucleic acid. Here, the ligases links the free 5′end of the NA conjugate substrate with the 3′ end of a target nucleicacid. Several single-strand ligases are known which advantageouslyrequire no additional template nucleic acid strand in order to ligatethe two nucleic acids. RNA ligases in particular, in contrast to DNAligases, link nucleic acids fairly efficiently without a template (cf.:England, T. E.; Nature, 275, 560-561 (1978) or England, T. E.; Bruce, A.G.; Uhlenbeck, O. C. METHODS IN ENZYMOLOGY 65(1), 65-74 (1980) orRomaniuk, Paul J.; Uhlenbeck, Olke C. Methods Enzymol. 100, 52-9(1983)). Thus, a RNA ligase may use a conjugate of a synthetic bindingunit (SBU) and a short modified nucleic acid section (NA) (as little asone nucleotide), and link the conjugate with the 3′ end of an RNA. Thisis reaction is preferred for providing enzymatically processedconjugates for the above-described applications without using a templatenucleic acid. In a variant of this embodiment, template dependentligases which carry out a ligation of two nucleic acids are used.

In another embodiment, target nucleic acids which have been hybridizedto the nucleic acid section of an NA/SBU conjugate are specificallydegraded by the addition of nucleases. In this embodiment, particularpreference is given to the use of restriction endonucleases and RNase H(which is double-strand specific). In these embodiments, either thenucleic acid (NA) of an NA/SBU conjugate or a target nucleic acid aredegraded, cut, hydrolyzed or fragmented by the nuclease. Again, theenzyme recognizes as a substrate an NA/SBU conjugate hybridized with atarget nucleic acid. For example, RNase H digests an RNA strand if thisstrand hybridizes to a DNA strand or an SBU-modified DNA strand (DNA/SBUconjugate). Such reactions are important, e.g., in the selectivedestruction of specific interfering RNAs present in a biological sample.In this example, a sample or cell containing the target RNA is mixedwith a DNA/SBU conjugate whose DNA sequence is complementary to the RNA.RNase H may be added to the mixture, or the natural endogenous RNAse Hactivity may be used in the case of total cell extracts. Surprisingly,we can show that NA/SBU conjugates can be used in the methods withoutreduction in the enzymatic activity as compared with using unconjugatedDNA.

Preferably, the nucleic acid section (NA) of the NA/SBU conjugatescomprises at least one nucleotide, preferably 1 to 50 nucleotides. Forligation reactions, particularly preferred nucleic acid section lengthsare 1 to 20 nucleotides, more preferably from 1 to 10, and mostpreferably from 1 to 5 nucleic acids for ligation. Preferred nucleicacid lengths are 10 to 40 nucleotides, more preferably 17 to 30nucleotides, for polymerization and enzymatic nucleic acid degradationreactions, which require a specifically hybridized sequence.Surprisingly, applicants have found that the enzymatic activity mayoccur as near as less than 30, 20, 15, 10, 7, 5, or 2 nucleotides, oreven 1 nucleotide of the point of conjugation between the NA and theSBU.

The enzymatic reaction can take place here using NA/SBU conjugates insolution, or immobilized NA/SBU conjugates (e.g., as in the case ofenzymatic reporting by primer extension). The NA/SBU conjugates modifiedenzymatically in solution may then be immobilized on such surfaces buySAU sorting after completion of the enzymatic reaction. Theenzymatically obtained conjugates thus produced and immobilized can nowbe used to investigate samples in various assay formats as describedabove, or the enzymatic process itself may be utilized as part of theassay.

The synthetic binding units (SBU) themselves cannot be synthesized,amplified, modified, processed, ligated, fragmented or hydrolyzed byenzymes which are known from nucleic acid technology, such aspolymerases, ligases, nucleases, restriction enzymes. This property isparticularly advantageous when using conjugates of synthetic bindingunits and nucleic acids in enzymatic processes as described here, as theSBU is not modified, blocked, removed or processed by the enzymaticsteps normally necessary for processing the sample or the nucleic acid.Thus, the use of NA/SBU conjugates is thus advantageous for theproduction of amplified conjugates analogously to the processesdescribed in JP 03151900 and WO 93/25563.

EXAMPLES Example 1 Synthesis of Conjugates of Nucleic Acids andSynthetic Binding Systems

Nucleic acids, oligonucleotides, synthetic binding systems and/or NA/SBUconjugates may be prepared according to the solid phase method on anautomated synthesizer Expedite 8905 from Applied Biosystems. As utilizedin the following examples, nucleic acids or oligonucleotides or thecorresponding nucleic acid part of conjugates are synthesized usingcommercially available phosphoramidites including the reversephosphoramidites for 5′-3′ synthesis (Cruachem). For solid synthesis ofnucleic acids, the synthesis cycles and conditions suggested by themanufacturer of the equipment and/or amidite supplier were used. Thesame is true for the use of linking building blocks, linkers, modifiersor branching amidites (as available from Glenn Research or Chemgenes).

For pRNA synthetic binding systems, the conditions and monomersdescribed in the appropriate references are used. Although the basicmethod for solid-phase synthesis of the pRNA synthetic binding systemsis the same, important differences compared with standard DNA synthesisare the use of adapted synthesis cycles with longer coupling time, theuse of 6% dichloroacetic acid for detritylation and the use ofpyridinium hydrochloride as activator. For conjugates, preference isgiven to using the β-cyanoethyl protective group on the phosphoramiditein conjunction with the special deprotection protocol describedhereinafter. The monomers are dried beforehand in vacuo over potassiumhydroxide and employed as a 0.1 M solution in dry acetonitrile. As analternative to pRNA or pDNA synthesized by phosphoramidite chemistry,CNA with its amidic backbone may be synthesized according to the methodsdescribed in WO 99/15509, and utilized with the proper functional groupmodification in the coupling reactions below.

The chromatographic purification of the nucleic acids, synthetic bindingsystems and conjugates is carried out via RP-HPLC. Column: MerckLiChrospher RP 18 or Phenomex Luna Phenyl-Hexyl, 10 μM, analytical:4×250 mm, flow=1.0 ml/min, semi-preparative: 10×250, flow=3.0 ml/min;buffer: A: 0.1 M triethylammonium acetate (TEAA) pH=7.0 in water, B: 0.1M TEAA pH=7.0 in acetonitrile/water (95:5); gradient: 0% B to 100% B in100 min for analytical and preparative separations).

Electrospray mass spectra (ESI-MS) were recorded on a Finnigan LCQapparatus in negative ionization mode. MALDI mass spectra were recordedon an Applied Biosystems Voyager DEPro.

Surprisingly, applicants have found that synthetic binding systems andconjugates are obtained in particularly good yields and in particularlygood purity if a treatment with an alkylamine, such as diethylamine indichloromethane, is carried out before deprotection and removal from thesynthesis support.

-   -   General protocol for cyanoethyl deprotection of synthetic        binding systems and conjugates:

After solid phase synthesis according to the phosphoramidite method, thesupport is initially mixed with a 1.5% (w/v) solution of diethylamine indichloromethane and shaken at room temperature, in the dark, overnight(15 h). It is also possible to rinse the synthesis support with thesolution of reagents continuously instead of using a batch process. Thesolution is discarded and the support is washed with three portions ofeach of the following solvents: dichloromethane, acetone, water. Duringthis treatment, the target molecules remain immobilized on the support.They are removed and deprotected only in the following step.

General Protocol for the Deprotection and Removal from the SynthesisSupport of Synthetic Binding Systems and Conjugates (Hydrazinolysis):

The moist CPG support from the above protocol is mixed with 24% aqueoushydrazine hydrate and shaken at 4° C. for 18 h. Hydrazine is removed bysolid phase extraction using Sep-Pak C18 cartridges 0.5 g Waters, No.20515. For this purpose, the cartridge is activated by rinsing with 10ml of acetonitrile. The hydrazine solution, diluted with five times thevolume of triethylammonium bicarbonate buffer (TEAB) pH 7.0, is thenloaded onto the column and the product is bound. Hydrazine is removed bywashing with TEAB. The product is then diluted with TEAB/acetonitrile(1:2). Alternatively, hydrazine may be removed via RP-HPLC if ahydrazine-resistant column is used. Here, Poros R3 (Applied Biosystems)has proven useful. The elution conditions correspond to those describedabove. Product-containing fractions are combined and evaporated todryness in vacuo. Analysis and preparative purification are carried outvia RP-HPLC as described above. For pDNA and conjugates thereof, it isalso possible to use aqueous ammonia for deprotection under theconditions common in DNA synthesis chemistry (25% solution, 55° C.,overnight).

Example 1.1 Conjugates of^(4′) pRNA^(2′)-^(5′)DNA^(3′) (FIG. 1C; FIG.2C; FIG. 2F)

The synthesis was begun with a standard solid phase DNA synthesis in the3′-5′ direction to produce the desired DNA sequence. If a linking groupor branching group was desired, the appropriate synthesis buildingblocks were coupled to the 5′ end of the product under the conditionsindicated by the manufacturer. If the DNA was directly linked to pRNAthrough a phosphate, (FIG. 2C), no linker phosphoramidite was used.Next, the monomer building blocks of pRNA were coupled under the specialconditions described above so that a SBU binding address of the desiredlength and sequence was obtained. If a labeled conjugate was desired,marker phosphoramidites were coupled to the conjugate in an optionallast step, or utilized as an initial coupled reagent in the synthesis.Here, the fluorescent dye phosphoramidites Cy3 and Cy5 (AmershamPharmacia Biotech) were useful, but it is also possible to use any othersuitable phosphoramidite dyes derivatives known from DNA synthesis. Theconjugate was worked up, deprotected, isolated and purified as describedabove. Analogously, it is possible to prepare conjugates of pRNA andmodified nucleic acids (e.g. 2′-O-methyl RNA). For this,2′-O-methylphosphoramidites are coupled after the pRNA. The nucleic acidwas also occasionally phosphorylated at its 5′ end, as shown below. Forthis purpose, building blocks known to the skilled worker forintroducing phosphates were used.

Applying the general protocol led to the following conjugates(bold=pRNA; italics=DNA): IL4RP102a: ^(4′) TCCTGCATTC ^(2′)-^(5′) GCATAG AGG CAG [SEQ ID NO. 77] AAT AAC AGG ^(3′) retention time: 23.7 min;M calc.: 9697; M obs.: 9696 IL4RP103a: ^(4′) CTCTACGTCT ^(2′) ^(5′) GCATAG AGG CAG [SEQ ID NO. 78] AAT AAC AGG ^(3′) retention time: 22.2 min;M calc.: 9697; M obs.: 9696 IL4RP104a: ^(4′) CCTCGTACTT ^(2′) ^(5′) GCATAG AGG CAG [SEQ ID NO. 79] AAT AAC AGG ^(3′) retention time: 21.7 min;M calc.: 9697; M obs.: 9696 Feto1-102a: ^(4′) TCCTGCATTC ^(2′) ^(5′) AGAAAT CTC ACA [SEQ ID NO. 80] TGG ACA TCT TCA ^(3′) retention time: 22.2min; M calc.: 10770; M obs.: 10771 Feto2-102a: ^(4′) TCCTGCATTC ^(2′)^(5′) CCT GGG CTT TGC [SEQ ID NO. 81] AGC ACT TCT C ^(3′) retentiontime: 22.3 min; M calc.: 9830; M obs.: 9822

Example 1.2 Conjugates of5′DNA^(3′)-^(4′)pRNA^(2′) (FIG. 1A: FIG. 2A:FIG. 2D)

The synthesis was begun with a pRNA synthesis as described above,producing the desired pRNA SBU on the synthesis support. If a linkinggroup or branching group was desired, the appropriate synthesis buildingblocks were coupled to the product using conditions indicated by themanufacturer. As above, when pRNA was directly linked to DNA by aphosphate (FIG. 2A), no further linker was added. Standard DNAphosphoramidites and synthesis cycles were then used in order tosynthesize the DNA sequence desired. If a labeled conjugate was desired,marker phosphoramidites were be coupled to the conjugate as describedabove. The conjugate was worked up, deprotected, isolated and purifiedas described above. Analogously, conjugates of 2′O-methyl RNA and pRNAwere also obtained by using if 2′-O-methylphosphoramidites instead ofDNA phosphoramidites.

Applying the general protocol led to the following conjugates(bold=pRNA; italics=DNA); italics, underlined=2′-O-methyl RNA): IL4CS-102a: ^(5′)CCC CAG TGC TGG ^(3′4)TCCTGCATTC^(2′) [SEQ ID NO. 82]retention time: - min; M calc.: 6800; M obs.: 6801 IL6 CS-103a ^(5′)ATGCTAAAGGACGTCATTGCACAATCTTAA [SEQ ID NO. 83] ^(3′4) CTCTACGTCT ^(2′)retention time: 27 min; M calc.: 12391; M obs.: - IL4R CS-104a: ^(5′)GGAGT TTG TAC ATG CGG TGG AGC [SEQ ID NO. 84] ^(3′4)CCTCGTACTT^(2′)retention time: 27 min; M calc.: 10354; M obs.: 10353 P-OMeC-TAGGATT:phosphate-^(5′) C ^(3′4′) TAGGATT ^(2′) [SEQ ID NO. 85] retention time:39′ min; M calc.: 3154; M obs.: 3153

Example 1.3 Conjugates of^(3′)DNA^(5′)-^(4′)pRNA (FIG. 1B; FIG. 2B: FIG.2E)

This preferred embodiment was carried out analogously to the protocoldescribed in Example 1.2. The only difference was using for thesynthesis of the DNA part reverse phosphoramidites (Cruachem) whichallow assembly of the DNA chain in the 5′-3′ direction. As in Example1.2, this also led to a conjugate carrying a free 3′ end. If the 3′ endis needed, for example, for an enzymatic elongation reaction (PCR), amarker must not be attached to this phosphate. It is possible to preparelabeled conjugates with a free 3′ phosphate by starting synthesis with asupport-bound dye, or using labeled amidites which allow continuation ofthe synthesis of the molecule at the start of the solid phase synthesis.Such an amidite is Cy3 phosphoramidite (Amersham Pharmacia Biotech).

Applying the general protocol led to the following conjugates(bold=pRNA; italics=DNA): IL4RP104aCy3: ^(3′) GGA CAA TAA GAC GGA GATACG [SEQ ID NO. 86] ^(5′4) CCTCGTACTT ^(2′) retention time: 25 min; Mcalc.: 10221; M obs.: 10220

Example 1.4 pRNA-DNA Conjugate as an Example of Conjugating a PreformedSynthetic Binding System with Nucleic Acid

The modified DNA oligonucleotide IL4CsrU, 5′-d(CCC CAG TGC TGG)-rU-3′[SEQ ID NO. 87] (FIG. 5) was obtained by standard DNA synthesis on aribo-U CPG support and obtained from a commercial DNA oligo supplier(BioSpring, Frankfurt am Main). An aliquot of this nucleic acid wasdissolved in an appropriate amount of deionized water to obtain a 5 mMsolution. 20 μl (100 nmol) of the solution were introduced into a 1.5 mlEppendorf microreaction vessel. 1 μl of a 0.1 M aqueous sodium periodatesolution was added. The vessel was left standing at room temperature inthe dark for one hour. Excess periodate was then removed by adding 1 μlof a 0.5 M sodium sulfite solution. After 15 minutes, 10 μl of a 1 Msodium phosphate buffer pH 7.4 were added and the resulting solution wastransferred to a 1.5 ml Eppendorf microreaction vessel in which 8.4 nmolof a hydrazide-modified dye-labeled pRNA oligo, 4′hydrazide-TTACGGAT-Cy3 2′ [SEQ ID NO. 88], had been evaporated todryness beforehand. After mixing, the solution was left standing in thedark for one hour. Work-up: the product was purified by means ofRP-HPLC. According to HPLC, the yield is 84% based on hydrazide-pRNAoligo used. 4.1 nmol of conjugate were isolated, which corresponds to anisolated yield of 49% conjugate. The conjugate was characterized bymeans of MALDI-TOF MS: M calc.: 7210; M obs.: 7180 (broad signal);retention time: DNA: 20.4 min; hydrazide pRNA: 41.2 min; conjugate: 38.2min

All nucleic acids which contain a terminal cyclic cis-diol (e.g., RNA,aptamers and aptazymes,) can be conjugated by this method.Alternatively, a ribonucleoside may be incorporated into a DNA, PNA, orother nucleic acid terminus, or a SBU (e.g., pRNA) for use as a couplingmoiety.

Example 1.5 pRNA-DNA Conjugate as an Example of Conjugating a PreformedSynthetic Binding System with a Nucleic Acid

For this method, the DNA should contain a cis-diol, which may beprovided by synthesizing the nucleic acid on glyceryl supports, e.g.:Glen Research Corp., Sterling, Va., USA; catalog No. 20-2933-41 (FIG.6). The modified DNA oligonucleotide (e.g.: IL4CSGly, 5′-CCC CAG TGCTGG-Gly-3′) [SEQ ID NO. 89] was dissolved in water and reacted withsodium periodate in a 1.5 ml Eppendorf microreaction vessel. Excesssodium periodate was removed by adding sodium sulfite solution. Sodiumphosphate buffer pH 7.4 was added and the resulting solution is mixedwith a hydrazide-modified pRNA oligo (e.g.: 4′ hydrazide-TAGGCATT-Cy32′) [SEQ ID NO. 90] and sodium cyanoborohydride. After mixing, thesolution was left standing in the dark. The mixture was worked up byHPLC and the product was characterized by MALDI-TOF MS.

Example 1.6 Conjugation of a Nucleic Acid with a Plurality of SyntheticBinding Units

Branching building blocks for use in the solid-phase synthesis ofoligonucleotides are known from the literature (Shchepinov, M. S.;Udalova, I. A.; Bridgman, A. J.; Southern, E. M; Nucleic Acids Res.; 25,4447-4454 (1997)). Coupling of a triple-branching building block(Trebler Phosphoramidite, Glen Research catalog No. 10- 1922-90) andsubsequent coupling of a hydrazide phosphoramidite to the 5′ end of astill support-bound DNA oligonucleotide with the sequence 5′-CCC CAG TGCTGG-3′ [SEQ ID NO. 91] led to a nucleic acid containing three reactivehydrazide-precursor end groups. The building block was deprotected andworked up by standard methods, as described in Example 2.2. The productDNA oligonucleotide was, analogously to the method in Example 1.4,reacted with a pRNA synthetic binding unit carrying a ribonucleotidebuilding block on one end. The pRNA was obtained by synthesizing the SBUon a ribo-U support, and subsequent deprotection. Carrying out theconjugation reaction according to the above mentioned protocol providedthe desired product (FIG. 8).

Example 2.1 Production of 4′-Biotin-pRNA as an Example of SynthesizingSynthetic Address Units (FIG. 4A)

A 10mer pRNA address unit synthesized according to the methods describedwas first deprotected on the CPG support by removing its tritylprotective group by dichloroacetic acid. After activation and couplingof a biotin phosphoramidite (Cruachem) with DCI and subsequentoxidation, a biotin derivative was thus introduced via a phosphodiesterbond at the 4′ end of the pRNA address. Chromatographic purification andanalytical characterization are carried out by the protocols described.

EXAMPLES

Bio-102b Bio-GAATGCAGGA: [SEQ ID NO. 92] retention time: 25.4 min; Mcalc.: 3753; M: obs.: 3754 Bio-103b Bio-AGACGTAGAG: [SEQ ID NO. 93]retention time: 26.2 min; M calc.: 3753; M: obs.: 3754 Bio-104bBio-AAGTACGAGG: [SEQ ID NO. 94] retention time: 26.1 min; M calc.: 3753;M.: obs.: 3754

Other biotin-labeled pRNA SAUs utilizes, for example, in the surfaceplasmon resonance (SPR) experiments described in Example 8, below, wereprepared analogously to those shown above.

Example 2.2 Production of 4′ Tetrahydrazide-pRNA as an Example ofSynthesizing Synthetic Address Units (FIG. 4B)

A 10mer address unit prepared according to the pRNA synthesis protocolabove was first deprotected on the CPG support by removing its tritylprotective group by dichloroacetic acid. After activation and couplingof a symmetric branching phosphoramidite (ChemGenes) with pyridiniumhydrochloride and subsequent oxidation, a branching diol was introducedvia a phosphodiester bond at the 4′ end of the pRNA. Then, a diesterphosphoramidite was double-coupled to produce a branched tetraesterbinding address which, after cyanoethyl deprotection and subsequenthydrazinolysis, was directly reacted to give the tetrahydrazide.

Protocol for preparing the diester phosphoramidite diethyl5-[[[[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]oxy]methyl]-1,3-benzenedicarboxylate:1.29 g (5 mmol) of diethyl-5-(hydroxymethyl)isophthalate[252.27] (98%,Aldrich; CAS 181425-91-2) were dissolved in 20 ml of dry dichloromethaneat room temperature and mixed with 2.59 g (40 mmol, 4 eq) ofN-ethyldiisopropylamine[129.25] and 1.3 g (11 mmol, 1.1 eq) of2-cyanoethyl N,N-diisopropylchlorophosphoramidite[236.68] (Aldrich; CAS89992-70-1). After 15 min at room temperature, thin layer chromatography(ethyl acetate/n-heptane (1:4) showed completion of the reaction. Themixture was concentrated and mixed with 30 ml of ethyl acetate/n-heptane(2:3). The precipitated hydrochloride was removed by filtration. Thefiltrate was concentrated and applied directly to a chromatographycolumn. Elution with ethyl acetate/n-heptane (1:4) gave 1.6 g (70%) ofdiethyl5-[[[[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]oxy]methyl]-1,3-benzenedicarboxylateas a colorless oil. C₂₂H₃₃N₂O₆P; ¹H-NMR 8.59 (m, 1 H, arom.), 8.21 (m, 2arom.), 4.87-4.75 (m, 2 H, CH₂ cyanoethyl), 4.41 (q, J=6.98 Hz, 4 H, CH₂ethyl), 3.95-3.80 (m, 2 H, 2'CH i-Pr), 3.74-3.61 (m, 2 H, CH₂cyanoethyl), 2.66 (t, J (P,H)=6.45 Hz, 2 H O—CH₂-arom), 1.41 (t, J=6 H,2×CH₃ ethyl), 1.23-1.20 (m, 12 H, CH₃, i-Pr); ³¹P-NMR (CDCl₃): δ=149.94;¹³C-NMR (CDCl₃): δ=165.8 (C═O), 140.2 (C—CH₂—O—P), 132.1 (2×C arom.),131.1 (2×C—H arom), 129.7 (C H arom), 117.6 (CN), 64.7 (P—O—CH₂-arom),61.4 (2×CH₂ ethyl), 58.6 (O—CH₂—CH₂—CN), 43.4 (2×C—H i-Pr), 24.7 (4×CH₃i-Pr), 20.5 (O—CH₂—CH₂—CN), 14.4 (CH₃ ethyl); HRMS 453.2156([M+H]+C₂₂H₃₄N₂O₆P calculated: 453.21545). Chromatographic purificationand analytical characterization were carried out according to theprotocols described above.

EXAMPLES

4HY-102b 4HY-GAATGCAGGA: [SEQ ID NO. 95] retention time: 20.7 min; Mcalc.: 3985; M: obs.: 3986 4HY-103b 4HY-AGACGTAGAG: [SEQ ID NO. 96]retention time: 20.8 min; M calc.: 3985; M: obs.: 3986 4HY-104b4HY-AAGTACGAGG: [SEQ ID NO. 97] retention time: 20.8 min; M calc.: 3985;M: obs.: 3986

Example 3.1 Immobilization of Hydrazide-Modified SAUs on ActiveElectronic Arrays

Experiments were carried out on a NanoChip™ Molecular BiologyWorkstation (Nanogen Inc. San Diego, USA). The immobilization ofhydrazide-modified SAUs uses arrays carrying a chemical modificationwhich can react with the hydrazide. A array modification for such use isan activated ester-modified permeation layer array as described in(PCT/US00/22205). Here, the surface of the support material was coatedwith polyacrylamide into which N-hydroxysuccinimide activated estershave been copolymerized. Immobilization efficiency was improved by usingSAUs carrying on one end several (e.g. four) hydrazides (see Example2.2). The hydrazide-modified SAUs were dissolved in 50 mM histidinebuffer at a concentration of 500 nM and electronically addressed to thedesired locations on the device at 2.2 V for 3 minutes. They wereattached at the locations via by reacting the hydrazides with theactivated esters to form covalent bonds.

The arrays obtained in this way were used for the immobilization ofconjugates in the same manner as the arrays having SAUs fixed via anon-covalent biotin/streptavidin interaction.

Example 3.2 Immobilization of Biotinylated Synthetic Address Units onActive Electronic Arrays

Experiments are carried out on a NanoChip™ Molecular Biology Workstationand NanoChip™ cartridges (Nanogen Inc. San Diego, USA). Theimmobilization of the synthetic address units on the array used standardelectronic addressing protocols as are stated by the manufacturer forDNA. Briefly, biotinylated pRNA address units were dissolved in 50 mMβ-histidine at a concentration of 500 nM. This solution was applied tothe chip and the address units were directed electronically to thedesired locations. For this purpose, the desired array locations arebiased at an electric potential which leads to concentration of theSAUs. For negatively charged synthetic address units, e.g., pRNAs, thepositions were biased positive. A good addressing procedure for pRNA andpDNA was addressing with +2.0 V for 1 minute, in the experience of theapplicants.

The array of synthetic address units obtained in this way, or by thehydrazide attachment method above, can be used universally. Depending onthe set of conjugates applied, such an array becomes a specialapplication-based array.

Example 4 Selective Immobilization of 10 Different Conjugates on ActiveElectronic Arrays

From the synthetic binding systems described in Example 8, 10 (N=10)systems were selected, which were substantially orthogonal, and do notsignificantly interact with one another (102a,b; 103a,b; 104a,b; 106a,b;109a,b; 110a,b; 117a,b; 119a,b; 120a,b; 122a,b). The immobilizationschedule and addressing schedule are depicted in the table below.

The synthetic address units were attached to locations on a NanoChip™,as in Example 3.2, by means of biotin-streptavidin. The 10 differentaddress units (“b” series) were loaded row by row such that identicaladdress units were fixed at each of the 10 locations in one row. Inorder to ensure that each of the 10 address units is loaded only ontoone row, the 10 active SAU attachment steps were carried outsuccessively, with rinsing between the steps. 1 2 3 4 5 6 7 8 9 10 1

2

3

4

5

6

NA

7

8

9

10

The complementary binding units used were the 10 pRNA sequences (‘a’series) complementary to the 10 address units. For detection on thearray, the binding units were labeled at the 2′ end with a dye moiety(Cy3). The affinities of the synthetic binding systems were checkedusing a voltage of +2.0 V for in 180s. The concentration of theindividual binding units was in all cases 100 nM in 50 mM histidinebuffer. Each complementary binding unit was actively addressed column bycolumn so that it was possible to study the binding of one binding unitto all 10 address units with one electrical activation. Using 10 activeaddressing steps successively (M=10), it was thus possible to test 10synthetic binding units for their pairing properties with the 10 addressunits. The time for this parallel assay was 2 hours, while assaying 10pairs on the surface plasmon resonance system (Example 8) required 50hours. Here, the time advantage of encoding realized by the possibleparallelization of working steps becomes immediately obvious. Afteractively directing the complementary binding addresses, the chip waswashed with 40 ml of 50 mM NaCl HBS buffer and then read by the Nanogenworkstation reader. The fluorograph obtained is shown in FIG. 15. Onlythe desired pairings show intense fluorescence. The fields with theheavy border in the table in FIG. 15 represent those positions of theNanogen chip on which synthetic binding systems are located in which theattached SAUs and the addressed SBUs correspond to each other.

Example 5 Immobilization of Capture/Stabilizer Oligonucleotides onActive Electronic Arrays via Synthetic Binding Systems

One hundred different capture/stabilizer oligonucleotides areimmobilized on an active electronic array as follows: first an array offixed SAUs is generated using 10 different SAUs (N=10). 10 arraylocations in columns (cf. FIG. 12) are in each case provided withidentical SAUs. The preparation of such an array is described in Example3.1 and Example 3.2.

Then, the conjugates of capture/stabilizer oligonucleotides and SBUs areprepared, as described in Example 1. Each oligonucleotide carries aspecific SBU address. The conjugates are combined to sets of 10conjugates each (in total 10 sets, M=10), each SBU having a particularNA conjugated to it in each set. The sets are dissolved in 50 mMhistidine buffer at a concentration of 10 nM (per conjugate) anddirected electronically to a first set of 10 positions. Care is takenhere that each SAU appears only once in the set of addressed locations.An easy method of assuring this is, as depicted in FIG. 12, by attachingthe SAUs in columns and addressing the sets of conjugates in rows.Electronic directing takes place at +2.1 V for 120 seconds. The sets maybe directed to the positions either sequentially, partly in parallel, orsimultaneously for all positions of the set. The specific recognition ofSAU and SBU immobilizes only the conjugate with the matching recognitionsequence on each individual location: in other words the set is sortedinto individual conjugates and the individual conjugates are immobilizedon specific array locations. Then the array is washed with 50 mMhistidine buffer and the next set of conjugates is applied. Again,directing to 10 positions and washing is carried out. After 10 cycles(M=10), 100 positions are occupied by different capture/stabilizeroligonucleotides, and the array can be used for hybridization or otherassays. Compared with sequentially loading an array with 100 individualnucleic acids, which requires 100 cycles, the number of cycles isreduced by 90%.

Using the nucleic acid array obtained in this way, it is possible tocarry out all experiments and assays which can be carried out using anarray prepared via direct immobilization of nucleic acids on the arraypositions, such as gene expression, SNP, and STR assays.

Example 5.2 Parallel Immobilization of Mixtures of pRNA-DNA Conjugates:Selectivity of Immobilization

On an active electronic array 5 pRNA SAUs (“b” Series, sequences 102b,103b, 104b, 106b, 109b) were attached in rows using biotin/streptavidininteractions as described previously (Example 2.1). A standardStreptavidin/Agarose NanoChip cartridge is used. Attaching conditions:50 mM Histidine 500 nM SAU, 2.0 V, 60 s.

Five different mixtures of 10 pRNA-DNA conjugates were made by mixingindividually synthesized conjugates (Example 1.2) at 50 nM each. Withineach mixture 9 conjugates were Cy3 labeled and one was unlabeled. Eachmixture was addressed simultaneously to a column of locations with fivedifferent pRNA addresses. Addressing conditions were 50 mM Histidine, 50nM for each conjugate, 2.0 V, 180 s.

-   -   Mixture 1: DTD-102a; EH1-103a-Cy3; CYP19-104a-Cy3;        178850-106a-Cy3; 192610-109a-Cy3; 195088-110a-Cy3;        EPHX2-117a-Cy3; NAT12-119a-Cy3; GSTA1-120a-Cy3; NAT1B-122a-Cy3    -   Mixture 2: DTD-102a-Cy3; EH1-103a; CYP19-104a-Cy3;        178850-106a-Cy3; 192610-109a-Cy3; 195088-110a-Cy3;        EPHX2-117a-Cy3; NAT12-119a-Cy3; GSTA1-120a-Cy3; NAT1B-122a-Cy3    -   Mixture 3: DTD-102a-Cy3; EH1-103a-Cy3; CYP19-104a;        178850-106a-Cy3; 192610-109a-Cy3; 195088-110a-Cy3;        EPHX2-117a-Cy3; NAT12-119a-Cy3; GSTA1-120a-Cy3; NAT1B-122a-Cy3    -   Mixture 4: DTD-102a-Cy3; EH1-103a-Cy3; CYP19-104a-Cy3;        178850-106a; 192610-109a-Cy3; 195088-110a-Cy3; EPHX2-117a-Cy3;        NAT12-119a-Cy3; GSTA1-120a-Cy3; NAT1B-122a-Cy3    -   Mixture 5: DTD-102a-Cy3; EH1-103a-Cy3; CYP19-104a-Cy3;        178850-106a-Cy3; 192610-109a; 195088-110a-Cy3; EPHX2-117a-Cy3;        NAT12-119a-Cy3; GSTA1-120a-Cy3; NAT1B-122a-Cy3

Unbound material was washed off and the Cy3 fluorescence of the array isimaged on an NanoChip Molecular Biology Workstation. The results arelisted in the following Table. (See FIG. 18). The one pad per columnwhich corresponds to the SAU/SBU of the unlabeled conjugate within themixture showed low fluorescence: This demonstrated that there was littleunspecific binding of the 9 other conjugates to these positions. 1 2 3 45 102b 97.2 1048.6 1048.6 1048.6 1048.6 103b 1048.6 211.1 1048.6 1048.61048.6 104b 1048.6 1048.6 233.0 1048.6 1048.6 106b 1048.6 1048.6 1048.6171.3 1048.6 109b 1048.6 1048.6 1048.6 1048.6 179.6Note:A fluorescence value of 1048.6 indicates saturated fluorescence. Thereal value is higher then.

As a second step, a mixture of Cy5 labeled DNA strands complementary tothe DNA part of the conjugates (Cy5-DTD-Comp, Cy5-EH1-Comp;Cy5-CYP19-Comp, CyS-178850-Comp; Cy5-192610-Comp.) was addressedsimultaneously to the columns of immobilized conjugates (25 nM per DNA,2.0 V, 180 s). Again Unbound material was washed off and the Cy5fluorescence was imaged. Fluorescence signals at all positions showedthat conjugates capture complementary DNA. The pads with unlabeledconjugates also captured DNA complements, demonstrating that the labelsdid not have any effect on hybridization efficiency. 1 2 3 4 5 102b1048.6 1048.6 1048.6 1048.6 1048.6 103b 724.6 663.4 631.1 868.7 1048.6104b 666.0 611.3 525.1 552.9 557.4 106b 611.2 692.6 488.1 540.4 682.6109b 541.6 614.7 552.4 611.1 668.9Note:A fluorescence value of 1048.6 indicates saturated fluorescence. Thereal value is higher then.

Sequences: Sequence italics = DNA (5#-3′); Name bold = pRNA (4′-2′) 102bBio-4′GAATGCAGGA2′ [SEQ ID NO. 98] 103b Bio-4′AGACGTAGAG2′ [SEQ ID NO.99] 104b Bio-4′AAGTACGAGG2′ [SEQ ID NO. 100] 106b Bio-4′AGGCATAAAG2′[SEQ ID NO. 101] 109b Bio-4′AACAAGTGGG2′ [SEQ ID NO. 102] DTD-102a5′CTCAACTGACATATAGCATTGGGCA3′- [SEQ ID NO. 103] 4′TCCTGCATTC2′ EH1-103a5′ACCCTCACTTCAAGACTAAGATTGAAGGTA3′- [SEQ ID NO. 104] 4′CTCTACGTCT2′CYP19-104a 5′ACCCGGTTGTAGTAGTTGCAGGCAC3′- [SEQ ID NO. 105]4′CCTCGTACTT2′ 178850-106a 5′ACAACAATTTGAAGCTTCTGTAATTTTG3′- [SEQ ID NO.106] 4′CTTTATGCCT2′ 192610-109a 5′TGTCAGCCTTGCTACTTGAAGGTAC3′- [SEQ IDNO. 107] 4′CCCACTTGTT2′ 195088-110a 5′CCCCTGTAGGTTGCTTAAAAGGGAC3′- [SEQID NO. 108] 4′TCTGCTCATC2′ EPHX2-117a 5′-GCATGGATGGCAGCATTGTTCTGAA-3′-[SEQ ID NO. 109] 4′TTCTATACTC2′ NAT12-119a5′-GAGGTTCAAGCGTAAATAAGTATATTT-3′- [SEQ ID NO. 110] 4′TGGTCGGTTG2′GSTA1-120a 5′-CTTCTTTCAGTGGGAGGGAACTATTGAG-3′- [SEQ ID NO. 111]4′TGGTTATCTG2′ NAT1B-122a 5′-ACATTTATTATTATTATTATTATTATTATTTG-3′- [SEQID NO. 112] 4′CTCCATGTTC2′ DTD-102a-Cy3 5′CTCAACTGACATATAGCATTGGGCA3′-[SEQ ID NO. 113] 4′TCCTGCATTC2′-Cy3 EH1-103a-Cy35′ACCCTCACTTCAAGACTAAGATTGAAGGTA3′- [SEQ ID NO. 114] 4′CTCTACGTCT2′-Cy3CYP19-104a-Cy3 5′ACCCGGTTGTAGTAGTTGCAGGCAC3′- [SEQ ID NO. 115]4′CCTCGTACTT2′-Cy3 178850-106a-Cy3 5′ACAACAATTTGAAGCTTCTGTAATTTTG3′-[SEQ ID NO. 116] 4′CTTTATGCCT2′-Cy3 192610-109a-Cy35′TGTCAGCCTTGCTACTTGAAGGTAC3′- [SEQ ID NO. 117] 4′CCCACTTGTT2′-Cy3195088-110a-Cy3 5′CCCCTGTAGGTTGCTTAAAAGGGAC3′- [SEQ ID NO. 118]4′TCTGCTCATC2′-Cy3 EPHX2-117a-Cy3 5′-GCATGGATGGCAGCATTGTTCTGAA-3′- [SEQID NO. 119] 4′TTCTATACTC2′-Cy3 NAT12-119a-Cy35′-GAGGTTCAAGCGTAAATAAGTATATTT-3′- [SEQ ID NO. 120] 4′TGGTCGGTTG2′-Cy3GSTA1-120a-Cy3 5′-CTTCTTTCAGTGGGAGGGAACTATTGAG-3′- [SEQ ID NO. 121]4′TGGTTATCTG2′-Cy3 NAT1B-122a-Cy35′-ACATTTATTATTATTATTATTATTATTATTTG-3′- [SEQ ID NO. 122]4′CTCCATGTTC2′-Cy3 Cy5-DTD-Comp 5′-Cy5-TGCCCAATGCTATATGTCAGTTGAG-3′ [SEQID NO. 123] Cy5-EH1-Comp 5′-Cy5-TACCTTCAATCTTAGTCTTGAAGTGAGGGT-3′ [SEQID NO. 124] Cy5-CYP19-Comp 5′-Cy5-GTGCCTGCAACTACTACAACCGGGT-3′ [SEQ IDNO. 125] Cy5-178850-Comp 5′-Cy5-CAAAATTACAGAAGCTTCAAATTGTTGT-3′ [SEQ IDNO. 126] Cy5-192610-Comp 5′-Cy5-GTACCTTCAAGTAGCAAGGCTGACA-3′ [SEQ ID NO.127] Cy5-195088-Comp 5′-Cy5-GTCCCTTTTAAGCAACCTACAGGGG-3′ [SEQ ID NO.128] Cy5-EPHX2-Comp 5′-Cy5-TTCAGAACAATGCTGCCATCCATGC-3′ [SEQ ID NO. 129]Cy5-NAT12-Comp. 5′-Cy5-AAATATACTTATTTACGCTTGAACCTC-3′ [SEQ ID NO. 130]Cy5-GSTA1-Comp. 5′-Cy5-CTC AAT AGT TCC CTC CCA CTG AAA [SEQ ID NO. 131]GAA G-3′ Cy5-NAT1B-Comp. 5′-Cy5-CAA ATA ATA ATA ATA ATA ATA ATA [SEQ IDNO. 132] ATA AAT GT-3′

Example 5.3 Parallel Immobilization of Mixtures of P-RNA-DNA Conjugates:Selectivity of Immobilization with 10 SAU/SBU

Step 1: P-RNA Synthetic Address Unit (SAU) Array Construction

On an active electronic array 10 p-RNA SAU's (“b” Series, sequences102b, 103b, 104b, 106b, 109b, 110b, 117b, 119b, 120b, 122b) wereattached in rows by biotin/streptavidin attachment (Example 2.1). Astandard Streptavidin/Agarose NanoChip cartridge was used. Attachingconditions: 50 mM Histidine 250 nM SAU, 2.0 V, 60 s. An array with 10identical p-RNA addresses in each row, each row having a distinct p-RNAaddress, was obtained.

Step 2: DNA Array Construction

One mixture of 10 p-RNA-DNA conjugates was made by mixing individuallysynthesized conjugates (Example 1.2) at 40 nM each. Within the conjugatemixture each DNA sequence was coded by a specific p-RNA SBU (DTD-102a,EH1-103a, CYP19-104a, 178850-106a, 192610-109a, 195088-110a, EPHX2-117a,NAT12-119a, GSTA1-120a, NAT1B-122a). The conjugate mixture was activelyaddressed simultaneously to all 10 pads of one column, with a set of 10locations with 10 different p-RNA addresses. Ten columns were addressedsequentially with the same conjugate mixture. Note: by using 10different mixtures of DNA-p-RNA conjugates (wherein within each mixtureeach DNA is coded by a different p-RNA SBU but in different mixturesdifferent DNA's are coded with repeating p-RNA SBU's) instead of oneconjugate mixture it, is possible to construct an array of 100 differentDNA sequences with the same number of addressing steps and within thesame time as in this experiment. Addressing conditions were 50 mMHistidine, 40 nM for each conjugate, 2.0 V, 180 s) The conjugate mixturewas heated to 95° C. for 5 min and rapidly chilled on an ice bathimmediately prior addressing in order to disrupt secondary structuresand putative DNA-DNA interactions. The sequences of the conjugates inthe mixture were as follows: DTD-102a 5′CTCAACTGACATATAGCATTGGGCA3′-[SEQ ID NO. 133] 4′TCCTGCATTC2′ EH1-103a5′ACCCTCACTTCAAGACTAAGATTGAAGGTA3′- [SEQ ID NO. 134] 4′CTCTACGTCT2′CYP19-104a 5′ACCCGGTTGTAGTAGTTGCAGGCAC3′- [SEQ ID NO. 135]4′CCTCGTACTT2′ 178850-106a 5′ACAACAATTTGAAGCTTCTGTAATTTTG3′- [SEQ ID NO.136] 4′CTTTATGCCT2′ 192610-109a 5′TGTCAGCCTTGCTACTTGAAGGTAC3′- [SEQ IDNO. 137] 4′CCCACTTGTT2′ 195088-110a 5′CCCCTGTAGGTTGCTTAAAAGGGAC3′- [SEQID NO. 138] 4′TCTGCTCATC2′ EPHX2-117a 5′-GCATGGATGGCAGCATTGTTCTGAA-3′-[SEQ ID NO. 139] 4′TTCTATACTC2′ NAT12-119a5′-GAGGTTCAAGCGTAAATAAGTATATTT-3′- [SEQ ID NO. 140] 4′TGGTCGGTTG2′GSTA1-120a 5′-CTTCTTTCAGTGGGAGGGAACTATTGAG-3′- [SEQ ID NO. 141]4′TGGTTATCTG2′ NAT1B-122a 5′-ACATTTATTATTATTATTATTATTATTATTTG-3′- [SEQID NO. 142] 4′CTCCATGTTC2′(Bold: p-RNA; Italics: DNA)

An array of DNA captures immobilized on an active electronic NanogenChip was obtained. Within each row of the chip the same DNA capturesequence was immobilized.

Step 3: Probing

To show the selectivity of the immobilization of the DNA capturesequences the chip was probed with 10 mixtures of unlabeled, Cy3, andCy5 labeled DNA reporter sequences complementary to the DNA capturesequences immobilized by the SBSs.

The mixtures were as follows:

-   -   a) Within each mixture one DNA reporter was unlabeled (Mixture        No. 1: reporter No. one; mixture No. 2: reporter No. 2; . . . ;        mixture No. 10: reporter No. 10)

b) Within each mixture every second reporter sequence was labeled withthe same type of fluorescent dye, and adjacent reporters has differentdyes, within the mixtures. DTDcomp 5′-TGC CCA ATG CTA TAT GTC AGT TGAG-3′ [SEQ ID NO. 143] EH1comp 5′-TAC CTT CAA TCT TAG TCT TGA AGT GAGGGT-3′ [SEQ ID NO. 144] CYP19comp 5′-GTG CCT GCA ACT ACT ACA ACC GGGT-3′ [SEQ ID NO. 145] 178850comp 5′-CAA AAT TAC AGA AGC TTC AAA TTG TTGT-3′ [SEQ ID NO. 146] 192610comp 5′-GTA CCT TCA AGT AGC AAG GCT GAC A-3′[SEQ ID NO. 147] 195088comp 5′-GTC CCT TTT AAG CAA CCT ACA GGG G-3′ [SEQID NO. 148] EPHX2comp 5′-TTC AGA ACA ATG CTG CCA TCC ATG C-Cy3-3′ [SEQID NO. 149] NAT12comp 5′-AAA TAT ACT TAT TTA CGC TTG AAC CTC-3′ [SEQ IDNO. 150] GSTA1comp 5′-CTC AAT AGT TCC CTC CCA CTG AAA GAA G-3′ [SEQ IDNO. 151] NAT1Bcomp 5′-CAA ATA ATA ATA ATA ATA ATA ATA ATA AAT GT-3′ [SEQID NO. 152] DTDcomp-Cy3 5′-TGC CCA ATG CTA TAT GTC AGT TGA G-Cy3-3′ [SEQID NO. 153] EH1comp-Cy3 5′-TAC CTT CAA TCT TAG TCT TGA AGT GAGGGT-Cy3-3′ [SEQ ID NO. 154] CYP19comp-Cy3 5′-GTG CCT GCA ACT ACT ACA ACCGGG T-Cy3-3′ [SEQ ID NO. 155] 178850comp-Cy3 5′-CAA AAT TAC AGA AGC TTCAAA TTG TTG T-Cy3-3′ [SEQ ID NO. 156] 192610comp-Cy3 5′-GTA CCT TCA AGTAGC AAG GCT GAC A-Cy3-3′ [SEQ ID NO. 157] 195088comp-Cy3 5′-GTC CCT TTTAAG CAA CCT ACA GGG G-Cy3-3′ [SEQ ID NO. 158] EPHX2comp-Cy3 5′-TTC AGAACA ATG CTG CCA TCC ATG C-Cy3-3′ [SEQ ID NO. 159] NAT12comp-Cy3 5′-AAATAT ACT TAT TTA CGC TTG AAC CTC-Cy3-3′ [SEQ ID NO. 160] GSTA1Comp-Cy35′-CTC AAT AGT TCC CTC CCA CTG AAA GAA G-Cy3-3′ [SEQ ID NO. 161]NAT1Bcom-Cy3 5′-CAA ATA ATA ATA ATA ATA ATA ATA ATA AAT GT-Cy3-3′ [SEQID NO. 162] DTDcomp-Cy5 5′-Cy5-TGC CCA ATG CTA TAT GTC AGT TGA G-3′ [SEQID NO. 163] EH1comp-Cy5 5′-Cy5-TAC CTT CAA TCT TAG TCT TGA AGT GAGGGT-3′ [SEQ ID NO. 164] CYP19comp-Cy5 5′-Cy5-GTG CCT GCA ACT ACT ACA ACCGGG T-3′ [SEQ ID NO. 165] 178850comp-Cy5 5′Cy5-CAA AAT TAC AGA AGC TTCAAA TTG TTG T-3′ [SEQ ID NO. 166] 192610comp-Cy5 5′-Cy5-GTA CCT TCA AGTAGC AAG GCT GAC A-3′ [SEQ ID NO. 167] 195088comp-Cy5 5′-Cy5-GTC CCT TTTAAG CAA CCT ACA GGG G-3′ [SEQ ID NO. 168] EPHX2comp-Cy5 5′-Cy5-TTC AGAACA ATG CTG CCA TCC ATG C-3′ [SEQ ID NO. 169] NAT12comp-Cy5 5′-Cy5-AAATAT ACT TAT TTA CGC TTG AAC CTC-3′ [SEQ ID NO. 170] GSTA1Comp-Cy55′-Cy5-CTC AAT AGT TCC CTC CCA CTG AAA GAA G-3′ [SEQ ID NO. 171]Mixture 1: DTDcomp, EH1comp-Cy3, CYP19comp-Cy5, 178850comp-Cy3,192610comp-Cy5, 195088comp-Cy3, EPHX2comp-Cy5, NAT12comp-Cy3,GSTA1comp-Cy5, NAT1Bcomp-Cy3Mixture 2: DTDcomp-Cy3, EH1comp, CYP19comp-Cy3, 178850comp-Cy5,192610comp-Cy3, 195088comp-Cy5, EPHX2comp-Cy3, NAT12comp-Cy5,GSTA1comp-Cy3, NAT1Bcomp-Cy5Mixture 3: DTDcomp-Cy5, EH1comp-Cy3, CYP19comp, 178850comp-Cy3,192610comp-Cy5, 195088comp-Cy3, EPHX2comp-Cy5, NAT12comp-Cy3,GSTA1comp-Cy5, NAT1Bcomp-Cy3Mixture 4: DTDcomp-Cy3; EH1comp-Cy5, CYP19comp-Cy3, 178850comp,192610comp-Cy3, 195088comp-Cy5, EPHX2comp-Cy3, NAT12comp-Cy5,GSTA1comp-Cy3, NAT1Bcomp-Cy5Mixture 5: DTDcomp-Cy5, EH1comp-Cy3, CYP19comp-Cy5, 178850comp-Cy3,192610comp, 195088comp-Cy3, EPHX2comp-Cy5, NAT12comp-Cy3, GSTA1comp-Cy5,NAT1Bcomp-Cy3Mixture 6: DTDcomp-Cy3, EH1comp-Cy5, CYP19comp-Cy3, 178850comp-Cy5,192610comp-Cy3, 195088comp, EPHX2comp-Cy3, NAT12comp-Cy5, GSTA1comp-Cy3,NAT1Bcomp-Cy5Mixture 7: DTDcomp-Cy5, EH1comp-Cy3, CYP19comp-Cy5, 178850comp-Cy3,192610comp-Cy5, 195088comp-Cy3, EPHX2comp, NAT12comp-Cy3, GSTA1comp-Cy5,NAT1Bcomp-Cy3Mixture 8: DTDcomp-Cy3, EH1comp-Cy5, CYP19comp-Cy3, 178850comp-Cy5,192610comp-Cy3, 195088comp-Cy5, EPHX2comp-Cy3, NAT12comp, GSTA1comp-Cy3,NAT1Bcomp-Cy5Mixture 9: DTDcomp-Cy5, EH1comp-Cy3, CYP19comp-Cy5, 178850comp-Cy3,192610comp-Cy5, 195088comp-Cy3, EPHX2comp-Cy5, NAT12comp-Cy3, GSTA1comp,NAT1Bcomp-Cy3Mixture 10: DTDcomp-Cy3, EH1comp-Cy5, CYP19comp-Cy3, 178850comp-Cy5,192610comp-Cy3, 195088comp-Cy5, EPHX2comp-Cy3, NAT12comp-Cy5,GSTA1comp-Cy3, NAT1Bcomp

The reporter mixtures were addressed actively. All 10 pads of one columnrepresenting a set of 10 different DNA captures were addressed inparallel with one reporter mixture. Ten columns were addressedsequentially each with a different reporter mixture. Addressingconditions were 50 mM Histidine, 25 nM for each conjugate, 2.0 V, 180 s.The reporter mixture was heated to 95° C. for 5 min and rapidly chilledon an ice bath immediately prior to addressing in order to disruptsecondary structures and putative DNA-DNA interactions. The sequences ofthe reporters in the mixture were as noted in the above table. The chipwas washed with high salt buffer (50 mM phosphate, 500 mM NaCl; pH7.0;20 steps at 75 μl/s for 3 sec each) to remove unbound reporteroligonucleotides.

Thus, an array of bound reporter oligonucleotides where adjacentreporters are labeled with different fluorescent dyes was produced. Onthe positions of the diagonal (1,1,; 2,2, . . . 10,10) the reporter wasunlabeled.

Step 4: Fluorescent Readout

The chip is imaged at low gain (128 μs) in both Cy3 (red) and CyS(green) channel: Frame 1: GREEN (Cy3 Channel) rows columns 1 2 3 4 5 6 78 9 10 1 31 461 38 484 38 496 40 518 53 585 2 572 47 529 65 535 66 65669 783 63 3 58 267 87 269 98 294 89 298 101 341 4 553 74 656 84 643 83658 69 657 65 5 32 568 55 717 68 727 66 722 77 659 6 573 x 586 72 863 77859 89 826 81 7 39 352 62 448 77 500 67 525 72 400 8 466 75 641 115 722111 711 88 670 97 9 58 478 94 576 102 650 107 645 92 582 10 456 90 45593 488 99 527 107 559 63Note:Position 6-2 is the reference electrode and not used for addressing

Frame 1: RED (Cy5 Channel) Rows columns 1 2 3 4 5 6 7 8 9 10 1 14 15 58316 467 15 527 15 644 16 2 14 17 19 606 30 542 28 667 30 720 3 189 20 2134 156 36 158 42 177 36 4 15 414 22 18 18 415 25 448 28 490 5 336 19 35818 18 17 458 23 496 23 6 15 x 22 686 22 17 20 723 34 717 7 356 31 411 38484 29 22 28 484 34 8 20 468 41 528 46 542 32 27 26 497 9 331 35 411 34454 38 485 34 23 24 10 19 388 43 345 47 351 39 403 23 16Note:Position 6-2 is the reference electrode and not used for addressing

The fluorescent pattern matches the expected localization of thefluorescence on the array, given the addressing patterns used. Thisdemonstrates that:

-   -   1.) although mixtures of conjugates are addressed in parallel,        each the DNA capture sequences is immobilized to a specific pad        (location) on the array as defined by its p-RNA SBU/SAU        recognition    -   2.) although addressed in parallel, each DNA reporter        oligonucleotide is specifically bound by its matching DNA        capture.

Example 5.4 Use of Immobilized Nucleic Acids in a SNP Determination

As a demonstration of the practical application of the synthetic bindingsystem-immobilized nucleic acids to various assay formats, a singlenucleotide polymorphism (SNP) type experiment was carried out usingimmobilized pRNA-DNA conjugates on a NanoChip™ device. The SNPdiscrimination assay format is standard on the device, and thisparticular variant utilizes base-stacking probes to better distinguishbetween wild-type and mutant nucleic acids. Instead of PCR ampliconssynthetic oligonucleotides were used as targets. WT target (synthetic)5′-ggc gtt ttg caa aca tac ctt caa [SEQ ID NO. 180] tct tag tct tga agtgag ggt Gtc tgt tga gaa tct cca cct g-3′ Mutant target (synthetic)5′-ggc gtt ttg caa aca tac ctt caa [SEQ ID NO. 181] tct tag tct tga agtgag ggt Atc tgt tga gaa tct cca cct g-3′ Capture-Stabilizer 3′p-RNA-102a 5′-acc ctc act tca aga cta aga ttg [SEQ ID NO. 182] aaggta-3′4′-TCCTGCATTC-2′ WT reporter (5′ Cy3) 5′-tct caa cag aC-3′ [SEQ IDNO 183] Mutant reporter (5′ Cy5) 5′-tct caa cag aT-3′ [SEQ ID NO 184]

A p-RNA synthetic binding address (H4-102b from Example 2.2) was fixedon four positions of a NanoChip cartridge as described in Experiment3.1. A capture-stabilizer DNA-p-RNA conjugate was addressed to all fourpositions. The conjugate was bound to the surface by the specific p-RNAinteraction leading to a single stranded DNA capture-stabilizerimmobilized on the chip surface. Addressing conditions for the capturewere: 50 nM conjugate; 50 mM Histidine; 2.0 V, 120 s. A wild type targetsequence was addressed to position #1 of the chip (40 nM DNA, 50 mMHistidine; 2.0 V, 120 s). After washing of unbound material withhistidine buffer, a mutant SNP target with a single base mutation wasaddressed to position #2 of the chip under the same conditions. Toposition #3 a 1:1 mixture of WT and mutant target was addressed. Toposition #4 a 1:4 mixture of WT and mutant target is addressed.

The reporting is done by passively hybridizing a mixture of the Cy3labeled WT reporter and the Cy5 labeled mutant reporter to all positionsof the chip (at 500 nM reporter, 50 mM phosphate pH 7.5, 500 mM NaCl).After washing with high salt buffer (50 mM phosphate pH 7.5, 500 mMNaCl) the fluorescence of the four chip positions is measured in the Cy3and C5 channel. The Cy3 fluorescence is normalized (100% for the WT atposition #1)and the Cy5 signal of the mutant SNP target is normalized(100%. at position #2).

The following signals are measured: Position 1 2 3 4 Sample WT SNPWT/SNP 1:1 WT/SNP 1:4 Cy3 signal (1 = 100) 100 1 30 7 Cy5 signal (2 =100) 1 100 20 63

Example 6 Immobilization of Amplified Nucleic Acids

Amplified nucleic acids are immobilized by using conjugates of syntheticbinding units and amplified nucleic acids. One method of obtaining suchconjugates is by using conjugates of SBUs and nucleic acids as primersfor enzymatic amplification reactions, as described below. Typicalamplification reactions for nucleic acids are the polymerase chainreaction (PCR), or isothermal methods as described by Westin et al. (J.Clinical Microbiol 39 (3) 1097-1104 (2001)). If the primer used in suchmethods is a conjugate, a conjugate is obtained in which the amplifiednucleic acids are linked to an SBU.

Conjugates of this kind are immobilized on support materials by usingthe conjugates in the addressing protocols described above forcapture/stabilizer conjugates: after the amplification reaction, theconjugates are combined, as desired, to form sets in which each SBUoccurs only once. The conjugates are desalted (BioRad Bio-Spin columns)and diluted with 50 mM histidine buffer down to a final concentration of2.5 to 10 nM. The conjugates are directed to the desired locations on aNanogen instrument according to the protocols of the manufacturer (+2.0V; 3 minutes). The specific recognition and binding of SBU and SAUimmobilizes the conjugates so that, as a result, an array of amplifiednucleic acids is obtained. Using this array, it is possible to carry outall experiments and assays which can be carried out using an arrayprepared via direct immobilization of nucleic acids amplicons on thearray positions, such as gene expression and SNP assays.

Example 7 Immobilization of Amplified Nucleic Acids for Gene ExpressionStudies

The question of the relative frequency of particular nucleic acids, themRNA of genes, is central to gene expression studies. For this purpose,mRNAs are isolated from tissues or cells, and specific sections thereofare amplified. mRNA is isolated from tissues, as described in DNAMicroarrays—A Practical Approach (Schena, M; Oxford University Press,Oxford, 1999), and particular nucleic acid sequences (genes) areamplified with specific biotinylated primers. If conjugates of nucleicacids and SBUs are used as primers instead, an encoded amplificationproduct is obtained for the appropriate species. The amplificationreaction is carried out for each time, stage or population individuallyaccording to the protocol stated. Identical gene sequences but fromdifferent times, stages or populations, are encoded with different SBUs.When examining a plurality of sequences at the same time, the nucleicacids may be amplified simultaneously according to a multiplexamplification reaction. After amplification, the conjugates are combinedinto sets such that

-   -   a) all times, stages or populations of one species, which are to        be compared, are gathered in one set (with a different SBU in        each case) and that    -   b) each SBU occurs only once in any one set.

An array of attached synthetic address units (SAU) is prepared accordingto the methods described in Example 3.1 and 3.2 ( See FIG. 13B). On thisarray, the sets are directed to the locations of the SAUs complementaryto the SBUs contained in the set by applying a voltage (+2.0 V for 3min). In this connection, at least those locations which carry SAUscomplementary to the SBUs of one gene are addressed simultaneously. As aresult, the times, stages or populations of one gene, which are to becompared, may be immobilized under identical conditions. The ampliconsbound to the array positions are then detected by hybridization with alabeled nucleic acid complementary to the gene amplicon.

Example 8 Detection of Stability and Selectivity of the Binding ofSynthetic Binding Systems in a Multiplex SPR Experiment with 14Different pRNA Address Unit/Binding Unit Pairs

All SPR (surface plasmon resonance) experiments described were carriedout in a BlAcore 2000 instrument from BlAcore (Uppsala, Sweden), withBlAcore Control 3.1 software. The sensograms recorded were evaluated andprocessed using the BlAevaluation 3.1 software. The biotinylated pRNAaddress units were attached to the surface of BlAcore chips “Sensor ChipSA.” Each chip comprises four “channels” (fluidic conduits with thesensor surface) which are available both for immobilization and fordetection. The buffer system used was BlAcore standard buffer HBS-EP.Each sensor chip used underwent a cleaning process prior to binding ofthe address units so that subsequently a constant baseline was obtainedon all four channels.

In order to clean the Sensor Chip for optimal use in the experiments, aprotocol was developed comprising the following steps:

-   -   1. 10 min washing with HBS-EP buffer (5 μl/min)    -   2. 1 min injection of 10 mM NaOH (5 μl/min)    -   3. 5 min washing with HBS-EP buffer (5 μl/min)    -   4. repeating twice steps 1-3    -   Immobilization of the pRNA synthetic address units:

Biotinylated pRNA address units with “b” sequences with a 4′ biotin(produced as in Example 2.1) were fixed to the Sensor Chip surface usingprotocols provided by the manufacturer of the instrument. Each channel(position on the BIAcore chip) is thus provided with a defined addressunit. In order to ensure this, the attachment step had to be carried outsuccessively for the 4 channels. The binding efficiency of the surfacewas improved by dissolving the address units separately in 500 mM NaClHBS-EP (instead of Standard BIAcore buffer 150 mM NaCl HBS-EP). Theconcentration of the synthetic address units was always 100 nM.

Each channel was prepared at 295 K for two minutes at a flow rate of 5μl/min so that after immobilization approximately 700-800 RU (resonanceunits) of substance were bound. After binding, each channel was washedwith HBS-EP standard buffer for a further 10 min. Thus it was possibleto bind four different address units per chip. In order to assay 14different address units, therefore 4 chips had to be used (3 chips with4 address units and 1 chip with 2 address units). Each chip was alwaystreated with identical protocols and buffer solutions.

Binding of complementary binding units to the surfaces:

A complementary synthetic binding unit was injected into all fourchannels of a Sensor Chip so that it was possible, using one injectionof substrate, to test directly four synthetic address units for theiraffinities to the binding unit. Binding of the complementary bindingunits was studied in 150 mM NaCl HBS-EP buffer at a flow rate of 30μl/min at 295 K and an injection time of 2 min. The concentration of the14 complementary binding units was always 300 nM: the solutions wereprepared separately. After each injection, a washing step of 20 minfollowed in order to be able to estimate the strength ofbinding/affinity of the binding addresses. After binding of theindividual conjugates was tested, the conjugates were stripped off bydisrupting the SAU/SBU interaction with 15 mM NaOH. Subsequently, thechip was ready for another injection, i.e. the surface of the supportmaterial was regenerated and was available for further assays. Theprotocol was carried out 14 times on one chip in order to study eachsynthetic address unit for affinity to the synthetic binding units. Forall 4 chips, therefore, the protocol had to be run 64 times.

Binding protocol of a Sensor Chip on 4 channels:

-   -   1. 2 min injection of complementary binding unit ×(30 μl/min)*    -   2. 20 min washing with HBS-EP buffer (30 μl/min)    -   3. 1 min injection of 10 mM NaOH (5 μl/min)    -   4. 5 min washing with HBS-EP buffer (5 μl/min)    -   5. 1 min injection of 10 mM NaOH (5 μl/min)    -   6. 20 min washing with HBS-EP buffer (30 μl/min)    -   7. repeating 13× steps 1-6

The names of the synthetic binding unit and synthetic address unitsstudied for their affinity and specificity are indicated in FIG. 14. Thenumber denotes the name of the binding systems; ‘b’ in this experimentwas the synthetic address unit fixed to the surface of the Sensor Chip;‘a’ in this experiment was the synthetic binding unit whose binding tothe address unit was shown. The synthetic address units were synthesizedand purified as pRNA according to the methods described in Example 2.1.

The result of the SPR binding study is depicted in FIG. 14. The diagonalclearly indicates the specific binding of the synthetic binding systems.Note that off of the diagonal, most of the SBUs do not interact withnon-corresponding SAUs, or do so to an extend that is much less thantheir interaction with their corresponding SAU. These sequences are“orthogonal” to each other. Off of the diagonal, unwanted interactionsappear at a few positions, for example the 118 and 119 pairs. These twoSBS sequences are only slightly orthogonal to each other. Combinationsof synthetic address units and synthetic binding units showing suchcross-reactions can be avoided if required for a particular experimentor for the construction of a particular array.

Example 9 Use of pRNA-DNA Conjugates as Primers in a PCR Reaction

For the PCR reaction a set of two different pRNA-DNA conjugates was used(set 1 and set 2) which serve as primers during the amplificationreaction. The preparation of the primers by solid-phase synthesis isdescribed in Example 1.1. With the aid of these novel primers, a definedsequence section from the gene of the αfetoprotein from Mus musculus wasamplified and isolated. The cDNA of the gene was used here foramplification. The primer constructs had the following sequences:

Set1: Fetol-102a (rev. 1977-1953); [SEQ ID NO. 172] ^(4′) TCCTGCATTC^(2′5′) AGA AAT CTC ACA TGG ACA TCT TCA ^(3′) Fetol (for.; 1796-1820):[SEQ ID NO. 173] 5′-G TCT GTT TCA CAG AAG AGG GTC CAA-3′

Set2: Feto2-102a (for 1796-1820): [SEQ ID NO. 174] ^(4′) TCCTGCATTC^(2′5′) CCT GGG CTT TGC AGC ACT TCT C ^(′) Feto2 (for.; 1634-1659): [SEQID NO. 175] 5′-GA TCT GTG CCA AGC TCA GGG CAA AG-3′

The sequence of the α-fetoprotein gene cDNA, from which the sequenceswere amplified, is: [SEQ ID NO. 176] Mouse mRNA encodingalpha-fetoprotein (a fetal serum protein): tcccacttcc agcactgcctgcggtgaagg aacaagcagc catgaagtgg atcacacccg cttccctcat cctcctgctacatttcgctg cgtccaaagc attgcacgaa aatgagtttg ggatagcttc cacgttagattcctcccagt gcgtgacgga gaagaatgtg cttagcatag ctaccatcac ctttacccagtttgttccgg aagccaccga ggaggaagtg aacaaaatga ctagcgatgt gttggctgcaatgaagaaaa actctggcga tgggtgttta gaaagccagc tatctgtgtt tctggatgaaatttgccatg agacggaact ctctaacaag tatggactct caggctgctg cagccaaagtggagtggaaa gacatcagtg tctgctggca cgcaagaaga ctgctccggc ctctgtcccacccttccagt ttccagaacc tgccgagagt tgcaaagcac atgaagaaaa cagggcagtgttcatgaaca ggttcatcta tgaagtgtca aggaggaacc ccttcatgta tgccccagccattctgtcct tggctgctca gtacgacaag gtcgttctgg catgctgcaa agctgacaacaaggaggagt gcttccagac aaagagagca tccattgcaa aggaattaag agaaggaagcatgttaaatg agcatgtatg ttcagtgata agaaaatttg gatcccgaaa cctccaggcaacaaccatta ttaagctaag tcaaaagtta actgaagcaa attttactga gattcagaagctggccctgg atgtggctca catccacgag gagtgttgcc aaggaaactc gctggagtgtctgcaggatg gggaaaaagt catgacatat atatgttctc aacaaaatat tctgtcaagcaaaatagcag agtgctgcaa attacccatg atccaactag gcttctgcat aattcacgcagagaatggcg tcaaacctga aggcttatct ctaaatccaa gccagttttt gggagacagaaattttgccc aattttcttc agaggaaaaa atcatgttca tggcaagctt tcttcatgaatactcaagaa ctcaccccaa ccttcctgtc tcagtcattc taagaattgc taaaacgtaccaggaaatat tggagaagtg ttcccagtct ggaaatctac ctggatgtca ggacaatctggaagaagaat tgcagaaaca catcgaggag agccaggcac tgtccaagca aagctgcgctctctaccaga ccttaggaga ctacaaatta caaaatctgt tccttattgg ttacacgaggaaagcccctc agctgacctc agcagagctg atcgacctca ccgggaagat ggtgagcattgcctccacgt gctgccagct cagcgaggag aaatggtccg gctgtggtga gggaatggccgacattttca ttggacattt gtgtataagg aatgaagcaa gccctgtgaa ctctggtatcagccactgct gcaactcttc gtattccaac aggaggctat gcatcaccag ttttctgagggatgaaacct atgcccctcc cccattctct gaggataaat tcatcttcca caaggatctgtgccaagctc agggcaaagc cctacagacc atgaaacaag agcttctcat taacctggtgaagcaaaagc ctgaactgac agaggagcag ctggcggctg tcactgcaga tttctcgggccttttggaga agtgctgcaa agcccaggac caggaagtct gtttcacaga agagggtccaaagttgattt ccaaaactcg tgatgctttg ggcgtttaaa catctccaga+E aggaagagtggacaaaaaaa tgtgttgacg ctttggtgtg agccttttgg cttaactgta actgctagtactttaaccac atggtgaaga+E tgtccatgtg agatttcta+E t accttaggaa taaaaacttttcaactatt

The amplified gene fragment to be expected using primer set 1 isunderlined in the sequence.

-   -   Amplification protocol:

For the amplification, 4 μl of a dNTP mix (2.5 mM; Promega), 2.5 μl ofFetol-102a (rev.) (10 μM), 2.5 μl of Fetol (for.) (10 μM), 1 μl oftaq-polymerase (5 U/μl; Promega) and 2 μl of isolated cDNA from Musmusculus were added to 30 μl of DEPC water (Ambion) 5 μl of 10×thermophilic DNA-polybuffer (Promega), 3 μl of MgCl₂ (25 mM; Promega).For the PCR reaction, the 50 μl mixture was subjected to the followingamplification cycle: pre-incubation at 95° C. for 2 min; 35-cycle PCR at94° C. (15 sec), 55° C. (30 sec), 72° C. (30 sec) and completion of theamplification, the reaction mixture was removed and stored at 4° C.

The amplification was carried out for different stages of expression ofthe α-fetoprotein gene using primer sets 1 and 2. Thus, the expressionof the murine α-fetoprotein gene in murine fetal liver was determinedfor day 8 and day 14 developmental stages using the novel primers. As acontrol, a parallel reaction was performed using standard DNA primers,and analyzed on an agarose gel (see FIG. 22) for comparison. Foranalysis of the gene expression, a sample (20 μl) was removed from thePCR mixture, mixed with 2 μl of 6× loading buffer (Sigma) and the bandswere separated on a 1.7% agarose gel.

As shown in FIG. 22, in Feto 1 and Feto 2 specific amplificationproducts of the gene are formed using the conjugate primers whenever theproducts are also obtained with DNA primers. Conversely, the conjugateprimers produce no product whenever the DNA primers produce no product(e.g., Feto 2 d8), showing that non-specific amplification is notoccurring. The dilution series of Feto 1-102a (rev.), in which theconjugate primer was used at decreasing concentrations, shows thatprimer dimers do not appear if relatively low amounts of the primer areused.

In addition, the SBU conjugate products of the PCR reaction were able tobe immobilized by SBUs on a NanoChip™, in a procedure analogous to thatin Example 4.

Example 10 Use of the pRNA-DNA Conjugates in Midi-Scale PCR (500 μlReaction)

For particular applications larger amounts of encoded amplified nucleicacids are required. The following example describes the use of thepRNA-DNA conjugate for primer amplification of a fragment of the murineα-fetoprotein gene from a cDNA library in a relatively large PCR mixture(500 μl). Using identical primer sets, as described in Example 9, thefollowing amplification protocol was applied.

For the amplification 4 μl of a dNTP mix (2.5 mM; Promega), 25 μl ofFetol-102a (rev.) (10 μM), 25 μl of Fetol (for.) (10 μM), 10 μl oftaq-polymerase (5 U/μl; Promega) and 20 μl of cDNA library from Musmusculus were added to 336 μl of DEPC water (Ambion) 50 μl of 10×thermophilic DNA-polybuffer (Promega), 30 μl of MgCl₂ (25 mM; Promega).For the PCR reaction, the 500 μl mixture was subjected to the followingamplification cycle: pre-incubation at 95° C. for 2 min; 35-cycle PCR at94° C. (15 sec), 55° C. (30 sec), 72° C. (30 sec) and finally 72° C. (7min). After completion of the amplification, the reaction mixture wasremoved and stored at 4° C.

Analogously to Example 9, an agarose gel electrophoresis was carried outfor the fractionation. The result is depicted in FIG. 22B. The genefragments obtained were purified with the aid of the Quiagenpurification kit and subsequently sequenced. The amplification of thedesired sequence was confirmed.

Example 11 Ligation of a Conjugate with a Nucleic Acid by Means of T4RNA Ligase

The enzymatic coupling between 5′-phosphate-(3′-O-methyl-RNA)-pRNAhybrid molecules (donor) and RNA (acceptor) by means of T4 RNA ligase isdescribed below. For background on ligation reactions, see England, T.E.; Nature, 275, 560-561 (1978) or England, T. E.; Bruce, A. G.;Uhlenbeck, O. C. Methods in Enzymology 65(1), 65-74 (1980) or Romaniuk,Paul J.; Uhlenbeck, Olke C. Methods Enzymol. 100, 52-9 (1983)). In thisreaction, a phosphodiester bond between the 5′ phosphate of the RNA-pRNAhybrid molecule and the 3′ OH group of an acceptor RNA is formed. Theacceptor RNA used was prepared by in-vitro transcription according froma DNA template, using a 5′ T7 promoter and T7 RNA polymerase.

Preparation of an in-vitro transcription template (for generating anacceptor RNA) by PCR using the following primers: Oligonucleotide #1,Primer forward: [SEQ ID NO. 177] 5′-TAATACGACTCACTATAGGG-3′Oligonucleotide #2, primer reverse: [SEQ ID NO. 178]5′-TGGGGCTAAGCGGGATCG-3′ Oligonucleotide #3, template: [SEQ ID NO. 179]5′-GCTGCAGTAATACGACTCACTATAGGGGCTATAGCTCAGCTGGGAGAGCGCTTGCCTGGGAAGCAAGAGGTCAGCGGTTCGATCCCGCTTAGCCCCA CCGCGGCGTCCATCCA-3′

A PCR reaction mixture consisting of 1 μM primer forward(oligonucleotide #1), 1 μM primer reverse (oligonucleotide #2), 0.2 μMtemplate (oligonucleotide #3), 2 mM MgCl₂, 50 mM KCl, 10 mM Tris/HCl (pH9.0 at 25° C.), 0.1% (v/v) TritonX-100, 0.2 mM dNTP, 0.05 U/μl Taqpolymerase (Promega) was used for a PCR amplification over 20 cyclesaccording to the following temperature program: 60 sec 95° C. 30 sec 95°C. 30 sec 53° C. {close oversize brace} 20 cycles 60 sec 72° C. 120 sec72° C.

The acceptor RNA was then prepared by in-vitro transcription from thetemplate. The unpurified PCR product was used as DNA template to anin-vitro transcription kit (Promega Ribomax™ largescale RNA productionsystem T7). The in-vitro transcription mixture was incubated at 37° C.for 2-6 h. Subsequently, the transcription products were purified eitherby a preparative urea gel or by an RNeasy™ purification column (Qiagen).The success of the transcription reaction was monitored by fractionatingthe transcription products on a 10% polyacrylamide urea gel.

Enzymatic Linkage Between RNA-pRNA Hybrid Molecules and Acceptor RNA byMeans of T4 RNA Ligase:

For ligating a 5′-phosphate-(3′-O-methyl-RNA)-pRNA-hybrid molecule(donor) with the acceptor RNA, 100 pmol of purified acceptor RNA wereincubated together with 300 pmol or 1000 pmol of a5′-phosphate-(3′-O-methyl-RNA)-pRNA-hybrid molecule (Described inExample 1.2) and 10 U T4 RNA ligase (MBI fermentas), 50 mM HEPES/NaOH(pH 8.0 at 25° C.), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 20 μg/ml BSA and10% (v/v) DMSO in a final reaction volume of 15 μl at 16° C. for 30 min.

To check the success of the ligation, an equal volume of a loadingbuffer (8.3 M urea, 50 mM EDTA) was added to the mixtures which, afterheat denaturation (5 min at 80° C.), were fractionated on a 10%polyacrylamide urea gel (40 mA for approx. 20 min). The nucleic acidswere then visualized in the gel by UV shadowing (FIG. 23A). In addition,it was possible to detect the particular ligation product in the gel bymeans of fluorescence scanning, owing to labeling the(3′-O-methyl-RNA)-pRNA half of the molecule with the fluorescence dyeCy3 (FIG. 23B). Since the acceptor RNA contains no dye, the RNA is notdetectable in the fluorescence image. Due to its short length, the freepRNA conjugate also does not appear in the fluorescence image.

Example 12 Use of SBU-NA Conjugates as Primers in Anchored SDA Reactions

SDA forward and reverse primers (each containing a BsoBI endonucleaserecognition sequence 5′ of a sequence which is specific for a targetnucleic acid) may be attached to SBUs by the methods described inExample 1. These are then electronically address to individual locationson the microchip array. Single or multiple SAU/SBU pairs can be employedat any one location, for the amplification of a single product, ormultiplex amplification. An exemplary target for amplification would bethe α-fetoprotein of Mus musculus described above, for which the aboveprimer sequences, may be utilized with the addition of a BsoBIrecognition site sequence (C/YCGRG). Template DNA from a sample is thenelectronically addressed and hybridized to the immobilized primers.

The amplification reaction is initiated by the addition of enzymes andbumper primers (Which hybridize 5′ to the SDA primers on each strand ofthe target nucleic acid.) The chip is warmed to 60° C. for 5 min. 10 μLof a pre-warmed SDA mixture (6 mM morpholinopropane sulfonic acid[MOPS]., pH 7.8; 1.7 mM [each] dGTP, dTTP, DATP, and thiolated dCTP; 85mM KCl, 18 mM MgCl₂; 23 mM NaCl; 3.5 mM Tris-HCl, pH 7.9; 35 μMdithiothreitol; 1.5 U BsoBI; 0.8 U BstI polymerase; and 25 nM eachbumper primer) is introduced to the chip. Following incubation for 30min. at 60° C., the amplification reaction is stopped by washing thechip 5 times with 37.5 mM NaCl, 3.75 mM sodium citrate, pH 7.2. Theanchored amplicons are denatured with alkaline solution (75 mM NaCl, 7mM sodium citrate, pH 12.5) for 4 min and washed 5 times with 37.5 mMNaCl, 3.75 mM sodium citrate, pH 7.2, to removed non-anchoredcomplementary amplicons. The anchored amplicons are subsequentlydetected via the hybridization with the appropriate reporter probe.

1. A method for producing an array of immobilized nucleic acids on anarray device, the method comprising the steps of: providing an arraydevice comprising a plurality of microlocations, wherein at least onemicrolocation comprises an electrode and a synthetic addressing unitcoupled to the at least one microlocation; activating the at least onemicrolocation; contacting the at least one microlocation with aconjugate, wherein the conjugate comprises a nucleic acid and asynthetic binding unit and wherein the synthetic binding unit is capableof binding to the synthetic addressing unit; and coupling the conjugateto the microlocation through binding of the synthetic binding unit tothe synthetic addressing unit.
 2. The method of claim 1, wherein thesynthetic addressing unit is coupled to at least two microlocations. 3.The method of claim 1, wherein the at least one microlocation isactivated by electronic biasing of the electrode.
 4. The method of claim1, further comprising the step of biasing the at least one microlocationto remove unbound conjugate.
 5. The method of claim 1, wherein thesynthetic binding unit is selected from the group consisting of pRNA,pDNA, and CNA.
 6. The method of claim 1, wherein the syntheticaddressing unit is selected from the group consisting of pRNA, pDNA, andCNA.
 7. The method of claim 1, wherein the synthetic binding unit isconnected via the a linkage between the 4′ end of the synthetic bindingunit to the 5′ end of the nucleic acid.
 8. The method of claim 1,wherein the synthetic binding unit is connected via the a linkagebetween the 2′ end of the synthetic binding unit to the 3′ end of thenucleic acid.
 9. The method of claim 1, wherein the synthetic bindingunit is connected via the a linkage between the 4′ end of the syntheticbinding unit to the 3′ end of the nucleic acid.
 10. The method of claim1, wherein the synthetic binding unit is connected via the a linkagebetween the 4′ end of the synthetic binding unit to the 5′ end of thenucleic acid.
 11. The method of claim 1, wherein the nucleic acid isselected from the group consisting of deoxyribonucleic acids,ribonucleic acids, and chemically modified nucleic acids.
 12. The methodof claim 1, wherein the nucleic acid is selected from the groupconsisting of phosphorothioate nucleic acids, phosphorodithioate nucleicacids, methylphosphonate nucleic acids, 2′-O-methyl RNA, and 2′-fluoroRNA.
 13. The method of claim 1, wherein the nucleic acid is selectedfrom the group consisting of peptide nucleic acids (PNA) and lockednucleic acids (LNA).
 14. The method of claim 1, wherein the conjugatesfurther comprise at least one labeling moiety.
 15. The method of claim14, wherein the labeling moiety is selected from the group consisting offluorescent moieties, quencher moieties, visible dye moieties,radioactive moieties, chemiluminescent moieties, biotin moieties, haptenmoieties, micro-particles, paramagnetic micro-particles, and enzymaticlabeling moieties.
 16. The method of claim 1, wherein the syntheticaddressing unit is coupled to the at least one microlocation via abiotin/streptavidin interaction.
 17. The method of claim 1, wherein thesynthetic addressing unit coupled to the at least one microlocation viaa covalent bond.
 18. The method of claim 1, wherein the at least onemicrolocation comprises an additional synthetic binding unit coupled tothe at least one microlocation.
 19. The method of claim 1, wherein thesynthetic addressing unit and synthetic binding unit are bound throughnon-covalent interaction.
 20. The method of claim 19, wherein thenon-covalent interaction is hydrogen bonding.